U.S. patent number 6,955,811 [Application Number 09/917,278] was granted by the patent office on 2005-10-18 for methods of inhibiting immune response suppression by administering antibodies to ox-2.
This patent grant is currently assigned to Trillium Therapeutics Inc.. Invention is credited to David A. Clark, Reginald M. Gorczynski.
United States Patent |
6,955,811 |
Gorczynski , et al. |
October 18, 2005 |
Methods of inhibiting immune response suppression by administering
antibodies to OX-2
Abstract
Methods and compositions for regulating immunity are disclosed.
For enhancing an immune response, agents that inhibit OX-2 are
administered. Such methods are useful in treating cancer. For
suppressing an immune response, an OX-2 protein or a nucleic acid
encoding an OX-2 protein is administered. Such methods are useful
in preventing graft rejection, fetal loss, autoimmune disease,
allergies and in inducing tumor cell growth.
Inventors: |
Gorczynski; Reginald M.
(Willowdale, CA), Clark; David A. (Burlington,
CA) |
Assignee: |
Trillium Therapeutics Inc.
(Toronto, CA)
|
Family
ID: |
46277925 |
Appl.
No.: |
09/917,278 |
Filed: |
July 30, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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570367 |
May 5, 2000 |
6338851 |
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PCTCA9801038 |
Nov 6, 1998 |
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Current U.S.
Class: |
424/154.1;
424/130.1; 530/388.1; 530/387.9; 530/387.3; 530/387.1; 424/144.1;
424/143.1; 424/142.1; 424/141.1; 424/133.1; 530/388.75;
530/388.22 |
Current CPC
Class: |
A61P
35/04 (20180101); A61P 35/00 (20180101); C07K
16/2803 (20130101); C07K 14/70503 (20130101); A61K
38/00 (20130101); C07K 2319/00 (20130101); C07K
2319/30 (20130101); C07K 2317/75 (20130101); C07K
2317/73 (20130101); A61K 2039/505 (20130101); A61K
48/00 (20130101); C07K 2317/76 (20130101) |
Current International
Class: |
C07K
14/435 (20060101); C07K 16/18 (20060101); C07K
14/705 (20060101); C07K 16/28 (20060101); A61K
48/00 (20060101); A61K 38/00 (20060101); A61K
039/395 (); C07K 016/28 (); C07K 016/30 () |
Field of
Search: |
;530/387.1,387.7,388.1,388.2,388.23,388.7,388.8,350
;424/130.1,138.1,139.1,141.1,143.1,152.1,153.1,155.1,172.1,173.1,174.1 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO 97/21450 |
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Jun 1997 |
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WO |
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WO 99/24565 |
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May 1999 |
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WO |
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Other References
Jain Nature Medicine 1998; 4(6):655-657. .
Kim et al. Cancer Res. 2001; 61:2031-2037. .
Kjaergaard et al. Cancer Res. 2000; 60:5514-5521. .
Boon Adv. Cancer Res. 1992; 58:177-210. .
Huang Pharmacol. Therapeutics 2000 86:201-215. .
Pardoll Clin. Immunol. 2000; 95(1):S44-S62. .
Auchincloss, H., Jr. 1995 Transplantation Immunology, 211-218.
.
Barclay, 1981, Immunology, 44, pp. 727-736. .
Barclay and Ward, 1982, European Journal Biochem, 129, pp. 447-458.
.
Borriello, F. et al., 1998, Mammalian Genome, Feb., 9(2), pp.
114-118. .
Borriello, F. et al., 1997, J. Immunol, 158, pp. 4549-4554. .
Chen, Z et al., Database Medline, 1997, Biochimica et Biophysica
Acta, Nov. 28, 1362(1), pp. 6-10. .
Clark DA, Manuel J, Gorczynski RM, Blajchman M. 2001. Labile CD200
tolerance signal important in transfusion-related immunomodulation
(TRIM) prevention of recurrent miscarriages. Amer. J. Reprod.
Immunol. 45;361. .
Gorczynski, R.M. et al., 1998, Transplantation, Apr. 27, 65(8), pp.
1106-1114. .
Gorczynski, R.M. et al., 1999, J. Immunol, 163:1654-1660. .
Preston, S. et al., 1997, European Journal of Immunology, vol. 27,
No. 8, pp. 1911-1918. .
Ni, J., et al. "An immunoadhesin incorporating the molecule OX-2 is
a potent immunosuppressant which prolongs allograft survival",
FASEB Journal, vol. 13, No. 5, p. A983 (1999). .
Ragheb, R. et al., "Preparation and functional properties of
monoclonal antibodies to human, mouse and rat OX-2", Immunology
Letters, vol. 68, No. 2-3, p. 311-315 (1999)..
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Primary Examiner: Gambel; Phillip
Assistant Examiner: Ouspenski; Ilia
Parent Case Text
This application is a continuation-in-part of U.S. application Ser.
No. 09/570,367 filed May 5, 1998 (now U.S. Pat. No. 6,338,851)
which is a continuation of PCT/CA98/01038 filed Nov. 6, 1998 (which
designated the U.S.) which claims the benefit of U.S. Provisional
application Ser. No. 60/064,764 filed Nov. 7, 1997 (now abandoned).
This application also claims benefit of U.S. Provisional
application Ser. No. 60/222,725 filed Aug. 3, 2000 (now pending).
All of the prior applications are incorporated herein in their
entirety.
Claims
We claim:
1. A method of inhibiting immune suppression caused by CD200 in an
animal in need thereof, comprising administering an effective
amount of anti-CD200 antibody that inhibits immune suppression by
CD200 to said animal.
2. The method according to claim 1, wherein the animal has cancer,
an infection, or acquired immune deficiency syndrome.
3. The method according to claim 1 wherein the animal has
cancer.
4. The method according to claim 1 wherein the animal is human.
5. The method according to claim 3 wherein the animal is human.
6. The method aocording to claim 4 wherein the antibody binds to a
human CD200 protein.
7. The method according to claim 5 wherein the antibody binds to a
human CD200 protein.
8. The method according to claim 3 wherein the cancer is a
hematopoietic cell cancer.
9. The method according to claim 8 wherein the hematopoietic cell
cancer is a leukemia.
Description
FIELD OF THE INVENTION
The present invention relates to methods and compositions for
modulating an immune response. The invention includes the use of
the protein OX-2 to enhance an immune response and to treat
cancer.
BACKGROUND OF THE INVENTION
The immune system protects the body from infectious agents and
disease and is critical to our survival. However, in certain
instances, the immune system can be the cause of illness. One
example is in autoimmune disease wherein the immune system attacks
its own host tissues, in many instances causing debilitating
illness and sometimes resulting in death. Examples of autoimmune
diseases include multiple sclerosis, type 1 insulin-dependent
diabetes mellitus, lupus erythematosus and arthritis. A second
example where the immune system can cause illness is during tissue
or organ transplantation. Except in the cases of genetically
identical animals, such as monozygotic twins, tissue and organ
transplants are rejected by the recipient's immune system as
foreign. The immune reaction against transplants is even more
pronounced in transplantation across species or
xenotransplantation. A third example where the immune system harms
the host is during an allergic reaction where the immune system is
activated by a generally innocuous antigen causing inflammation and
in some cases tissue damage.
In order to inhibit the detrimental immune reactions during
transplantation, autoimmune disease and allergic reactions,
immunosuppressive drugs (such as cyclosporin A, tacrolimas, and
corticosteroids) or antibody therapies (such as anti-T cell
antibodies) are generally administered. Unfortunately, these
non-specific modes of immunosuppression generally have undesirable
side effects. For example, cyclosporin may cause decreased renal
function, hypertension, toxicity and it must be administered for
the life of the patient. Corticosteroids may cause decreased
resistance to infection, painful arthritis, osteoporosis and
cataracts. The anti-T cell antibodies may cause fever,
hypertension, diarrhea or sterile meningitis and are quite
expensive.
In view of the problems associated with immunosuppression, there
has been an interest in developing methods or therapies that induce
unresponsiveness or tolerance in the host to a transplant, to
"self" tissues in autoimmune disease and to harmless antigens
associated with allergies. The inventor has been studying the
mechanisms involved in transplant rejection and has developed
methods for inducing a state of antigen-specific immunological
tolerance in transplantation. In particular, in animal allograft
models, the inventor has demonstrated that graft survival can been
increased if the recipient animal is given a pre-transplant
infusion via the portal vein of irradiated spleen cells from the
donor animal. In contrast, a pre-transplant infusion via the tail
vein does not prolong graft survival.
Understanding the molecular mechanisms involved in the induction of
tolerance following portal-venous (pv) immunization may lead to the
development of methods of modulating an immune response that may be
useful in suppressing an immune response (for example in treating
transplant rejection, autoimmune disease and allergies) and in
enhancing an immune response (for example in treating cancer and
infectious diseases).
SUMMARY OF THE INVENTION
Genes that show an increase in expression following portal venous
immunization have been identified. One of the genes isolated
encodes OX-2, a molecule with previously unknown function belonging
to the Ig superfamily. The OX-2 molecule is also generally referred
to as CD200 in the current literature. The inventors have shown
that administering antibodies to OX-2 inhibited the graft survival
generally seen following pre-transplant pv immunization. The
inventors have also shown that there is a negative association
between levels of OX-2 and risk of fetal loss. In particular, the
inventors have shown administering OX-2 reduced fetal loss rates
while inhibiting OX-2 reversed the effect. The inventors have
further shown that OX-2 inhibits cytotoxic cells and IL-2
production and induces IL-4 production. The inventors have also
shown that OX-2 is responsible for promoting tumor metastases and
inhibiting OX-2 reduces tumor cell growth. All of these results
demonstrate that OX-2 is involved in immune suppression.
Consequently, broadly stated, the present invention provides a
method of suppressing an immune response comprising administering
an effective amount of an OX-2 protein or a nucleic acid sequence
encoding an OX-2 protein to an animal in need of such
treatment.
In one embodiment, the present invention provides a method of
preventing or inhibiting fetal loss comprising administering an
effective amount of an OX-2 protein or a nucleic acid sequence
encoding an OX-2 protein to an animal in need thereof. In another
embodiment, the present invention provides a method of inducing
tumor cell growth or metastases comprising administering an
effective amount of an OX-2 protein or a nucleic acid sequence
encoding an OX-2 protein to an animal in need thereof.
The invention also includes pharmaceutical compositions containing
OX-2 proteins or nucleic acids encoding OX-2 proteins for use in
inducing tolerance in transplantation or autoimmune disease.
The inventors have shown that inhibiting OX-2 prevents immune
suppression and is useful in treating cancer and inducing fetal
loss.
Therefore, in another aspect, the present invention provides a
method of preventing immune suppression comprising administering an
effective amount of an agent that inhibits OX-2 to an animal in
need thereof. In a preferred embodiment the OX-2 inhibitor is an
antibody that binds OX-2 or an antisense oligonucleotide that
inhibits the expression of OX-2.
In one embodiment, the present invention provides a method of
inhibiting the growth of a tumor cell comprising administering an
effective amount of an agent that inhibits OX-2 to a cell or animal
in need thereof.
In one embodiment, the present invention provides a method of
inducing fetal loss comprising administering an effective amount of
an agent that inhibits OX-2 to an animal in need thereof.
The invention also includes pharmaceutical compositions containing
an OX-2 inhibitor for use in inducing or augmenting an immune
response.
Other features and advantages of the present invention will become
apparent from the following detailed description. It should be
understood, however, that the detailed description and the specific
examples while indicating preferred embodiments of the invention
are given by way of illustration only, since various changes and
modifications within the spirit and scope of the invention will
become apparent to those skilled in the art from this detailed
description.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention will now be described in relation to the drawings in
which:
FIG. 1 illustrates PCR validation of suppressive subtractive
hybridization using .beta.-actin primers.
FIG. 2 illustrates PCR validation of suppressive subtractive
hybridization using IL-10 primers.
FIG. 3 is an autoradiograph using .sup.32 P-labeled probes from 4
clones obtained from the subtractive hybridization process.
FIG. 4 is flow cytometry profile of spleen adherent cells.
FIGS. 5A and B are Western Blots illustrating the increased
expression of OX-2 antigen after pv immunization. FIG. 5A shows
staining with a control mouse antibody, anti-mouse CD8a. FIG. 5B
shows staining with anti-rat MRC OX-2.
FIG. 6 is a graft showing percent survival versus days post renal
transplantation.
FIG. 7 shows the cDNA sequence of rat (SEQ.ID.NO.:20), mouse
(SEQ.ID.NO.:22) and human MRC OX-2 (SEQ.ID.NO.:18).
FIG. 8 shows the deduced protein sequence of rat (SEQ.ID.NO.:21),
mouse (SEQ.ID.NO.:2) and human MRC OX-2 (SEQ.ID.NO.:19)
protein.
FIGS. 9A and 9B are bar graphs showing cytokine production and cell
proliferation following stimulation by allogeneic DC using hepatic
NPMC.
FIGS. 10A, 10B and 10C are bar graphs showing inhibition of cell
proliferation and cytokine production by hepatic NPMC.
FIG. 11 is a bar graph analysis of FACS data showing OX-2
expression in a subpopulation of NPC.
FIG. 12 shows PCR analysis mRNA expression of B7-1, B7-2 and OX-2
in various hepatic NPMC cell fractions.
FIGS. 13A and 13B are bar graphs showing proliferation and cytokine
production by NPMC from Flt3L treated mice.
FIG. 14 is a bar graph showing cytokines produced from C3H mice
with C57BL16 renal allografts and NPC from Flt3 treated C57BL16
donors.
FIG. 15 is a graph showing inhibition of graft rejection with NPC
from Flt3 treated mice.
FIG. 16 is a graph showing that anti-OX-2 reverses inhibition by
NPC. The effect of anti-B7-1, anti-B7-2 and anti-OX-2 on primary
allostimulation is shown.
FIG. 17 is a graph showing that anti-OX-2 mAb reverses inhibition
by NPC and inhibits the development of immunoregulatory cells.
FIG. 18A is a photograph showing in situ hybridization with
antisense OX-2 in a 8-11 day placenta from a mouse that has
undergone fetal loss.
FIG. 18B is a photograph showing in situ hybridization with
antisense OX-2 in a 8-11 day placenta from a mouse that is not
susceptible to spontaneous fetal loss.
FIG. 19 is a graph showing inhibition of EL4 or C1498 tumor growth
in C3H bone marrow reconstituted C57BL/6 mice. Groups of 6 BL/6
mice received 20.times.10.sup.6 T-depleted BL/6 or C3H bone marrow
cells 24 hrs following cyclophosphamide treatment. 5.times.10.sup.6
EL4 or 5.times.10.sup.5 C1498 tumor cells were injected 28 days
later into these mice, and control BL/6 or C3H mice. >85% of PBL
from C3H reconstituted BL/6 were stained by FITC anti-H2K.sup.k mAb
at this time.
FIG. 20 is a graph showing EL4 tumor growth in BL/6 mice immunized
twice, at 14 day intervals, with 5.times.10.sup.6 EL4 cells
transfected to express CD80 or CD86. 5.times.10.sup.6 EL4 cells
were injected as tumor challenge 10 days after the last
immunization.
FIG. 21 is a graph showing suppression of growth inhibition in
C57BL/6 BMT recipients of EL4 or C1498 tumor cells (see FIG. 19)
following 4 weekly infusions of 100 .mu.g/mouse anti-CD4 or
anti-CD8 mAb, beginning on the day of BMT (tumor cells were
injected at 28 days post BMT). Data are shown for 6 mice/group.
FIG. 22 is a graph showing inhibition of immunity to EL4 or C1498
tumor challenge following infusion of CD200Fc in C57BL/6 mice
reconstituted with C3H bone marrow--see FIG. 19 and text for more
details. Cyclophosphamide treated BL/6 mice received bone marrow
rescue with T-depleted C3H or BL/6 cells. 28 days later all mice,
and groups of control normal C3H mice, received ip injection with
5.times.10.sup.6 EL4 or 5.times.10.sup.5 C1498 tumor cells. Bone
marrow reconstituted mice received further iv infusion of normal
mouse IgG or CD200Fc (10 .mu.g/mouse/injection) 5 times at 2 day
intervals beginning on the day of tumor injection.
FIG. 23 is a graph showing inhibition of immunity to EL4 tumor
cells in EL4-CD80 immunized BL/6 mice using CD200Fc--see FIG. 20
and text for details. Mice received iv infusion of control IgG or
CD200Fc as described in FIG. 22.
FIG. 24 is a graph showing improved tumor immunity in EL4-CD86 or
C1498-CD86 immunized C57BL/6 mice following infusion of anti-CD200
mAb. See legend to FIG. 20 and text for more details. Where shown,
groups of mice received iv infusion of anti-CD200, 100 mg/mouse, on
3 occasions at 3 day intervals beginning on the day of tumor
injection.
FIG. 25 is a graph showing log10 relative concentrations of CD200
mRNAs compared with standardized control mRNA. All samples were
first normalized for equivalent concentrations of GAPDH mRNA.
Values shown represent arithmetic means .+-.SD for 3 individual
samples for each time point. Mice were preimmunized with
CD80/CD86-transfected tumor cells as described in the text.
FIG. 26 is a graph showing increased inhibition of tumor immunity
using infusion of CD200Fc with CD200.sup.r+ cells in C57BL/6
recipients of C3H bone marrow. In this experiment some mice
received not only CD200Fc with EL4 or C1498 tumor, but in addition
a lymphocyte-depleted, LPS-stimulated, macrophage population
stained (>65%) with anti-CD200.sup.r mAb (2F9).
FIG. 27 is a graph showing combinations of CD200Fc and anti-CD4 or
anti-CD8 mAb produce increased suppression of tumor growth
inhibition in C57BL/6 recipients of C3H BMT. Groups of 6 mice
received weekly iv infusions of 100 .mu.g anti-T cell mab or 5 iv
infusions of 10 .mu.g/mouse CD200Fc, alone or in combination,
beginning on the day of tumor injection (28 days post BMT).
FIG. 28 is a graph showing effect of combined CD200Fc and anti-CD4
or anti-CD8 mAb on suppression of EL4 tumor growth inhibition in
C57BL/6 recipients preimmunized with EL4-CD80 transfected cells
(see FIG. 20). Data are shown for groups of 6 mice/group. Weekly iv
infusions of 100 .mu.g anti-T cell mab or 5 iv infusions of 10
.mu.g/mouse CD200Fc, alone or in combination, were begun on the day
of tumor injection (10 days after the final immunization with
EL4-CD80 cells).
FIGS. 29A and B are bar graphs showing the median number of lung
nodules in mice receiving allogeneic blood by tail vein.
FIGS. 30A and B are bar graphs showing the number of lung nodules
in the presence of anti-OX2 in mice receiving allogeneic blood by
tail vein.
FIGS. 31A and B are bar graphs showing the number of lung nodules
in the presence of anti-OX-2, DEC205 or anti-CD11c in mice
receiving allogeneic blood by tail vein.
DETAILED DESCRIPTION OF THE INVENTION
The present inventors have identified genes that show an increase
in expression following portal venous immunization. These genes
play a role in the development of immune suppression or tolerance
and may be useful in developing therapies for the prevention and
treatment of transplant rejection, fetal loss, autoimmune disease
allergies, and cancer.
Using suppression subtractive hybridization (SSH), the inventor has
isolated a clone that is preferentially expressed in mice receiving
allogenic renal grafts along with pre-transplant donor-specific
immunization and that encodes the protein OX-2. The OX-2 protein
(also known as MRC OX-2) in rat was described as a 41Kd-47Kd
glycoprotein which is expressed on the cell surface of thymocytes,
follicular dendritic cells and endothelium, B cells and neuronal
cells. Differences in apparent size of the molecule in different
tissues is probably a function of differential glycosylation. The
function of the molecule was previously unknown, but DNA and amino
acid sequence analysis shows it has a high degree of homology to
molecules of the immunoglobulin gene family, which includes
molecules important in lymphocyte antigen recognition and cell-cell
interaction (e.g. CD4, CD8, ICAMs, VCAMs), as well as adhesion
receptor molecules (NCAMs) in the nervous system. Members of the
immunoglobulin superfamily are distinct from other molecules of the
integrin and selectin families, which, at least within the immune
system, also seem to play critical role in cell recognition,
migration and even development of the lymphocyte recognition
repertoire (by regulating intra-thymic selection events). It has
become increasingly evident that molecules of these different
families play an important role in human disease.
The inventors have shown that administering antibodies to OX-2
inhibited the graft survival generally seen following
pre-transplant pv immunization. The inventors have also shown that
there is negative association between levels of OX-2 and risk of
fetal loss and that administering OX-2 prevents fetal loss and
inhibiting OX-2 causes fetal loss. The inventors have further shown
that OX-2 promotes tumor cell growth and inhibiting OX-2 inhibits
tumor cell growth. The inventors have also shown that OX-2 inhibits
cytotoxic cells and IL-2 production and induces IL-4 production.
All of the data supports the role of OX-2 as a potent immune
regulator and that modulating OX-2 can be useful in suppressing or
enhancing an immune response.
Therapeutic Methods
(a) Preventing Immune Suppression
In one aspect, the present invention provides a method of
preventing immune suppression or enhancing an immune response by
administering an agent that inhibits OX-2 to an animal in need
thereof.
There are a large number of situations whereby it is desirable to
prevent immune suppression including, but not limited to, the
treatment of infections, cancer and Acquired Immune Deficiency
Syndrome and the induction of fetal loss.
Accordingly, the present invention provides a method of preventing
immune suppression comprising administering an effective amount of
an agent that inhibits OX-2 to an animal in need thereof.
The term "effective amount" as used herein means an amount
effective, at dosages and for periods of time necessary to achieve
the desired result such as preventing immune suppression.
The term "animal" includes all members of the animal kingdom and is
preferably a mammal, more preferably a human.
The agent that inhibits OX-2 can be any agent that decreases the
expression or activity of an OX-2 protein such that the immune
suppression caused by OX-2 is reduced, inhibited and/or prevented.
Such agents can be selected from agents that inhibit OX-2 activity
(such as antibodies, OX-2 ligands, small molecules), agents that
inhibit OX-2 expression (such as antisense molecules) or agents
that inhibit the interaction of OX-2 with its receptor (such as
soluble OX-2 receptor and antibodies that bind the OX-2
receptor).
One of skill in the art can readily determine whether or not a
particular agent is effective in inhibiting OX-2. For example, the
agent can be tested in in vitro assays to determine if the function
or activity of OX-2 is inhibited. The agent can also be tested for
its ability to induce an immune response using in vitro immune
assays including, but not limited to, enhancing a cytotoxic T cell
response; inducing interleukin-2 (IL-2) production; inducing
IFN.gamma. production; inducing a Th1 cytokine profile; inhibiting
IL-4 production; inhibiting TGF.beta. production; inhibiting IL-10
production; inhibiting a Th2 cytokine profile and any other assay
that would be known to one of skill in the art to be useful in
detecting immune activation.
In one embodiment the present invention provides a method of
inhibiting, preventing or reducing tumor cell growth comprising
administering an effective amount of an agent that inhibits OX-2 to
a cell or an animal in need thereof. Preferably, the animal is an
animal with cancer, more preferably human.
One of skill in the art can determine whether a particular agent is
useful in inhibiting tumor cell growth. As mentioned above, one can
test the agent for its ability to induce an immune response using
known in vitro assays. In addition, the agent can be tested in an
animal model, for example as described in Examples 8 and 9, wherein
the agent is administered to an animal with cancer.
The term "inhibiting or reducing tumor cell growth" means that the
agent that inhibits OX-2 causes an inhibition or reduction in the
growth or metastasis of a tumor as compared to the growth observed
in the absence of the agent. The agent may also be used
prophylactically to prevent the growth of tumor cells.
The tumor cell can be any type of cancer including, but not limited
to, hematopoietic cell cancers (including leukemias and lymphomas),
colon cancer, lung cancer, kidney cancer, pancreas cancer,
endometrial cancer, thyroid cancer, oral cancer, laryngeal cancer,
hepatocellular cancer, bile duct cancer, squamous cell carcinoma,
prostate cancer, breast cancer, cervical cancer, colorectal cancer,
melanomas. and any other tumors which are antigenic or weakly
antigenic. This could include, for example, EBV-induced neoplasms,
and neoplasms occurring in immunosuppressed pateints, e.g.
transplant patients, AIDS patients, etc.
(i) Antibodies
In a preferred embodiment, the agent that inhibits OX-2 is an OX-2
specific antibody. The present inventor has prepared antibodies to
OX-2 which are described in Examples 4 and 5. Antibodies to OX-2
may also be obtained commercially or prepared using techniques
known in the art such as those described by Kohler and Milstein,
Nature 256, 495 (1975) and in U.S. Pat. Nos. RE 32,011; 4,902,614;
4,543,439; and 4,411,993, which are incorporated herein by
reference. (See also Monoclonal Antibodies, Hybridomas: A New
Dimension in Biological Analyses, Plenum Press, Kennett, McKearn,
and Bechtol (eds.), 1980, and Antibodies: A Laboratory Manual,
Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988,
which are also incorporated herein by reference).
Conventional methods can be used to prepare the antibodies. For
example, by using the OX-2 protein, polyclonal antisera or
monoclonal antibodies can be made using standard methods. A mammal,
(e.g., a mouse, hamster, or rabbit) can be immunized with an
immunogenic form of the OX 2 protein which elicits an antibody
response in the mammal. Techniques for conferring immunogenicity on
a peptide include conjugation to carriers or other techniques well
known in the art. For example, the peptide can be administered in
the presence of adjuvant. The progress of immunization can be
monitored by detection of antibody titers in plasma or serum.
Standard ELISA or other immunoassay procedures can be used with the
immunogen as antigen to assess the levels of antibodies. Following
immunization, antisera can be obtained and, if desired, polyclonal
antibodies isolated from the sera.
To produce monoclonal antibodies, antibody producing cells
(lymphocytes) can be harvested from an immunized animal and fused
with myeloma cells by standard somatic cell fusion procedures thus
immortalizing these cells and yielding hybridoma cells. Such
techniques are well known in the art, (e.g., the hybridoma
technique originally developed by Kohler and Milstein (Nature 256,
495-497 (1975)) as well as other techniques such as the human
B-cell hybridoma technique (Kozbor et al., Immunol. Today 4, 72
(1983)); the EBV-hybridoma technique to produce human monoclonal
antibodies (Cole et al. Monoclonal Antibodies in Cancer Therapy
(1985) Allen R. Bliss, Inc., pages 77-96); and screening of
combinatorial antibody libraries (Huse et al., Science 246, 1275
(1989)). Hybridoma cells can be screened immunochemically for
production of antibodies specifically reactive with the OX-2
protein and the monoclonal antibodies can be isolated. Therefore,
the invention also contemplates hybridoma cells secreting
monoclonal antibodies with specificity for OX-2.
The term "antibody" as used herein is intended to include fragments
thereof which also specifically react with OX-2 or a peptide
thereof. Antibodies can be fragmented using conventional techniques
and the fragments screened for utility in the same manner as
described above. For example, F(ab').sub.2 fragments can be
generated by treating antibody with pepsin. The resulting F(ab')2
fragment can be treated to reduce disulfide bridges to produce Fab'
fragments.
Chimeric antibody derivatives, i.e., antibody molecules that
combine a non-human animal variable region and a human constant
region are also contemplated within the scope of the invention.
Chimeric antibody molecules can include, for example, the antigen
binding domain from an antibody of a mouse, rat, or other species,
with human constant regions. Conventional methods may be used to
make chimeric antibodies containing the immunoglobulin variable
region which recognizes an OX-2 protein (See, for example, Morrison
et al., Proc. Natl Acad. Sci. U.S.A. 81,6851 (1985); Takeda et al.,
Nature 314, 452 (1985), Cabilly et al., U.S. Pat. No. 4,816,567;
Boss et al., U.S. Pat. No. 4,816,397; Tanaguchi et al., European
Patent Publication EP171496; European Patent Publication 0173494,
United Kingdom patent GB 2177096B).
Monoclonal or chimeric antibodies specifically reactive with the
OX-2 as described herein can be further humanized by producing
human constant region chimeras, in which parts of the variable
regions, particularly the conserved framework regions of the
antigen-binding domain, are of human origin and only the
hypervariable regions are of non human origin. Such immunoglobulin
molecules may be made by techniques known in the art (e.g., Teng et
al., Proc. Natl. Acad. Sci. U.S.A., 80, 7308-7312 (1983); Kozbor et
al., Immunology Today, 4, 7279 (1983); Olsson et al., Meth.
Enzymol., 92, 3-16 (1982); and PCT Publication WO 92/06193 or EP
0239400). Humanized antibodies can also be commercially produced
(Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, Great
Britain.)
Specific antibodies, or antibody fragments reactive against OX-2
may also be generated by screening expression libraries encoding
immunoglobulin genes, or portions thereof, expressed in bacteria
with peptides produced from nucleic acid molecules of the present
invention. For example, complete Fab fragments, VH regions and FV
regions can be expressed in bacteria using phage expression
libraries (See for example Ward et al., Nature 341, 544-546:
(1989); Huse et al., Science 246, 1275-1281 (1989); and McCafferty
et al. Nature 348, 552-554 (1990)).
Accordingly, the present invention provides a method of preventing
immune suppression or inhibiting, preventing or reducing tumor cell
growth comprising administering an effective amount of an antibody
that inhibits OX-2 to an animal in need thereof. The invention also
includes the use of an antibody that inhibits OX-2 to prepare a
medicament to inhibit, prevent or reduce tumor cell growth.
(ii) Antisense Oligonucleotides
In another embodiment, the OX-2 inhibitor is an antisense
oligonucleotide that inhibits the expression of OX-2. Antisense
oligonucleotides that are complimentary to a nucleic acid sequence
from an OX-2 gene can be used in the methods of the present
invention to inhibit OX-2. The present inventors have prepared
antisense oligonucleotides to OX-2 which are described in Example
3.
Accordingly, the present invention provides a method of preventing
immune suppression or inhibiting tumor cell growth comprising
administering an effective amount of an antisense oligonucleotide
that is complimentary to a nucleic acid sequence from a OX-2 gene
to an animal in need thereof.
The term "antisense oligonucleotide" as used herein means a
nucleotide sequence that is complimentary to its target, the sense
strand of messenger RNA that is translated into protein at the
ribosomal level.
In one embodiment of the invention, the present invention provides
an antisense oligonucleotide that is complimentary to a nucleic
acid molecule having a sequence as shown in FIG. 7, wherein T can
also be U, or a fragment thereof.
The term "oligonucleotide" refers to an oligomer or polymer of
nucleotide or nucleoside monomers consisting of naturally occurring
bases, sugars, and intersugar (backbone) linkages. The term also
includes modified or substituted oligomers comprising non-naturally
occurring monomers or portions thereof, which function similarly.
Such modified or substituted oligonucleotides may be preferred over
naturally occurring forms because of properties such as enhanced
cellular uptake, or increased stability in the presence of
nucleases. The term also includes chimeric oligonucleotides which
contain two or more chemically distinct regions. For example,
chimeric oligonucleotides may contain at least one region of
modified nucleotides that confer beneficial properties (e.g.
increased nuclease resistance, increased uptake into cells), or two
or more oligonucleotides of the invention may be joined to form a
chimeric oligonucleotide.
The antisense oligonucleotides of the present invention may be
ribonucleic or deoxyribonucleic acids and may contain naturally
occurring bases including adenine, guanine, cytosine, thymidine and
uracil. The oligonucleotides may also contain modified bases such
as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and
other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil,
6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil,
8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl
adenines, 8-hydroxyl adenine and other 8-substituted adenines,
8-halo guanines, 8-amino guanine, 8-thiol guanine, 8-thiolalkyl
guanines, 8-hydroxyl guanine and other 8-substituted guanines,
other aza and deaza uracils, thymidines, cytosines, adenines, or
guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine.
Other antisense oligonucleotides of the invention may contain
modified phosphorous, oxygen heteroatoms in the phosphate backbone,
short chain alkyl or cycloalkyl intersugar linkages or short chain
heteroatomic or heterocyclic intersugar linkages. For example, the
antisense oligonucleotides may contain phosphorothioates,
phosphotriesters, methyl phosphonates, and phosphorodithioates. In
an embodiment of the invention there are phosphorothioate bonds
links between the four to six 3'-terminus bases. In another
embodiment phosphorothioate bonds link all the nucleotides.
The antisense oligonucleotides of the invention may also comprise
nucleotide analogs that may be better suited as therapeutic or
experimental reagents. An example of an oligonucleotide analogue is
a peptide nucleic acid (PNA) wherein the deoxyribose (or ribose)
phosphate backbone in the DNA (or RNA), is replaced with a
polyamide backbone which is similar to that found in peptides (P.
E. Nielsen, et al Science 1991, 254, 1497). PNA analogues have been
shown to be resistant to degradation by enzymes and to have
extended lives in vivo and in vitro. PNAs also bind stronger to a
complimentary DNA sequence due to the lack of charge repulsion
between the PNA strand and the DNA strand. Other oligonucleotides
may contain nucleotides containing polymer backbones, cyclic
backbones, or acyclic backbones. For example, the nucleotides may
have morpholino backbone structures (U.S. Pat. No. 5,034,506).
Oligonucleotides may also contain groups such as reporter groups, a
group for improving the pharmacokinetic properties of an
oligonucleotide, or a group for improving the pharmacodynamic
properties of an antisense oligonucleotide. Antisense
oligonucleotides may also have sugar mimetics.
The antisense nucleic acid molecules may be constructed using
chemical synthesis and enzymatic ligation reactions using
procedures known in the art. The antisense nucleic acid molecules
of the invention or a fragment thereof, may be chemically
synthesized using naturally occurring nucleotides or variously
modified nucleotides designed to increase the biological stability
of the molecules or to increase the physical stability of the
duplex formed with mRNA or the native gene e.g. phosphorothioate
derivatives and acridine substituted nucleotides. The antisense
sequences may be produced biologically using an expression vector
introduced into cells in the form of a recombinant plasmid,
phagemid or attenuated virus in which antisense sequences are
produced under the control of a high efficiency regulatory region,
the activity of which may be determined by the cell type into which
the vector is introduced.
(iii) Other OX-2 Inhibitors
In addition to antibodies and antisense molecules, other agents
that inhibit OX-2 may also be used in the present invention.
Accordingly, the present invention also includes the isolation of
other ligands or molecules that can bind to OX-2 or the OX-2
receptor. Biological samples and commercially available libraries
may be tested for proteins that bind to OX-2 or the OX-2 receptor.
In addition, antibodies prepared to the OX-2 or the OX-2 receptor
may be used to isolate other peptides with OX-2 or OX-2 receptor
binding affinity. For example, labelled antibodies may be used to
probe phage displays libraries or biological samples.
Conditions which permit the formation of protein complexes may be
selected having regard to factors such as the nature and amounts of
the substance and the protein.
The substance-protein complex, free substance or non-complexed
proteins may be isolated by conventional isolation techniques, for
example, salting out, chromatography, electrophoresis, gel
filtration, fractionation, absorption, polyacrylamide gel
electrophoresis, agglutination, or combinations thereof. To
facilitate the assay of the components, the antibodies, proteins,
or substances may be labelled with a detectable substance.
Once potential binding partners have been isolated, screening
methods may be designed in order to determine if the molecules that
bind to the OX-2 peptide or OX-2 receptor and are useful in the
methods of the present invention.
Therefore, the invention also provides methods for identifying
substances which are capable of binding to the OX-2. In particular,
the methods may be used to identify substances which are capable of
binding to and which suppress the effects of OX-2. Accordingly the
invention provides a method of identifying substances which bind
with OX-2, comprising the steps of:
(a) reacting OX-2 and a substance, under conditions which allow for
formation of a complex, and
(b) assaying for complexes, for free substance, and for non
complexed OX-2.
Substances which can bind with the OX-2 of the invention may be
identified by reacting OX-2 with a substance which potentially
binds to the OX-2, and assaying for complexes, for free substance,
or for non-complexed OX-2. Any assay system or testing method that
detects protein-protein interactions may be used including
co-immunoprecipitation, crosslinking and co-purification through
gradients or chromatographic columns may be used. Additionally,
x-ray crystallographic studies may be used as a means of evaluating
interactions with substances and molecules. For example, purified
recombinant molecules in a complex of the invention when
crystallized in a suitable form are amenable to detection of
intra-molecular interactions by x-ray crystallography. Spectroscopy
may also be used to detect interactions and in particular, Q-TOF
instrumentation may be used. Biological samples and commercially
available libraries may be tested for OX-2-binding peptides. In
addition, antibodies prepared to the peptides of the invention may
be used to isolate other peptides with OX-2 binding affinity. For
example, labelled antibodies may be used to probe phage display
libraries or biological samples. In this respect peptides of the
invention may be developed using a biological expression system.
The use of these systems allows the production of large libraries
of random peptide sequences and the screening of these libraries
for peptide sequences that bind to particular proteins. Libraries
may be produced by cloning synthetic DNA that encodes random
peptide sequences into appropriate expression vectors. (see
Christian et al. 1992, J. Mol. Biol. 227:711; Devlin et al., 1990
Science 249:404; Cwirla et al. 1990, Proc. Natl. Acad, Sci. USA,
87:6378). Libraries may also be constructed by concurrent synthesis
of overlapping peptides (see U.S. Pat. No. 4,708,871).
It will be understood that the agonist and antagonist that can be
assayed using the methods of the invention may act on one or more
of the binding sites on the protein or substance including agonist
binding sites, competitive antagonist binding sites,
non-competitive antagonist binding sites or allosteric sites.
The invention also makes it possible to screen for antagonists that
inhibit the effects of an agonist of the interaction of OX-2 with a
substance which is capable of binding to OX-2. Thus, the invention
may be used to assay for a substance that competes for the same
binding site of OX-2. As such it will also be appreciated that
intracellular substances which are capable of binding to OX-2 may
be identified using the methods described herein.
The reagents suitable for applying the methods of the invention to
evaluate substances and compounds that affect or modulate a OX-2
may be packaged into convenient kits providing the necessary
materials packaged into suitable containers. The kits may also
include suitable supports useful in performing the methods of the
invention.
(b) Inducing Immune Suppression
In another aspect, the present invention provides a method of
suppressing an immune response comprising administering an
effective amount of an OX-2 protein or a nucleic acid sequence
encoding an OX-2 protein to an animal in need of such treatment.
The invention includes a use of an effective amount of an OX-2
protein or a nucleic acid sequence encoding an OX-2 protein to
suppress an immune response.
The term "OX-2 protein" includes OX-2 or CD200 from any species or
source and includes a full length OX-2 protein as well as fragments
or portions of the protein. The term "OX-2" is also generally
referred to as "CD200" due to a change in nomenclature. Both "OX-2"
and "CD200" may be used interchangeably in the application.
Preferred fragments or portions of the OX-2 or CD200 protein are
those that are sufficient to suppress an immune response.
Determining whether a particular OX-2 or CD200 protein can suppress
an immune response can be assessing using known in vitro immune
assays including, but not limited to, inhibiting a mixed leucocyte
reaction; inhibiting a cytotoxic T cell response; inhibiting
interleukin-2 production; inhibiting IFN.gamma. production;
inhibiting a Th1 cytokine profile; inducing IL-4 production;
inducing TGF.beta. production; inducing IL-10 production; inducing
a Th2 cytokine profile; and any other assay that would be known to
one of skill in the art to be useful in detecting immune
suppression.
The term "administering an OX-2 protein" includes both the
administration of the OX-2 protein as well as the administration of
a nucleic acid sequence encoding an OX-2 protein. In the latter
case, the OX-2 protein is produced in vivo in the animal.
In a preferred embodiment, the OX-2 protein is prepared and
administered as a soluble fusion protein. The fusion protein may
contain the extracellular domain of OX-2 linked to an
immunoglobulin (Ig) Fc Region. The OX-2 fusion may be prepared
using techniques known in the art. Generally, a DNA sequence
encoding the extracellular domain of OX-2 is linked to a DNA
sequence encoding the Fc of the Ig and expressed in an appropriate
expression system where the OX-2- Fclg fusion protein is
produced.
The OX-2 or protein may be obtained from known sources or prepared
using recombinant DNA techniques. The protein may have any of the
known published sequences for OX-2 or CD200. The sequences can be
obtained from GenBank. The human sequence has accession no.
M17226.times.0523; the rat sequence has accession no. X01785; and
the mouse sequence has accession no. AF029214. The nucleic acid and
protein sequences of OX-2 (CD200) from human, mouse and rat are
also shown in FIGS. 7 and 8 and in SEQ. ID. Nos.: 18, 22 and 20
(nucleic acid) and SEQ. ID. Nos.:19, 21 and 2 (protein).
The OX-2 protein may also be modified to contain amino acid
substitutions, insertions and/or deletions that do not alter the
immunosuppressive properties of the protein. Conserved amino acid
substitutions involve replacing one or more amino acids of the OX-2
amino acid sequence with amino acids of similar charge, size,
and/or hydrophobicity characteristics. When only conserved
substitutions are made the resulting analog should be functionally
equivalent to the OX-2 protein. Non-conserved substitutions involve
replacing one or more amino acids of the OX-2 amino acid sequence
with one or more amino acids which possess dissimilar charge, size,
and/or hydrophobicity characteristics.
The OX-2 protein may be modified to make it more therapeutically
effective or suitable. For example, the OX-2 protein may be
cyclized as cyclization allows a peptide to assume a more
favourable conformation. Cyclization of the OX-2 peptides may be
achieved using techniques known in the art. In particular,
disulphide bonds may be formed between two appropriately spaced
components having free sulfhydryl groups. The bonds may be formed
between side chains of amino acids, non-amino acid components or a
combination of the two. In addition, the OX-2 protein or peptides
of the present invention may be converted into pharmaceutical salts
by reacting with inorganic acids including hydrochloric acid,
sulphuric acid, hydrobromic acid, phosphoric acid, etc., or organic
acids including formic acid, acetic acid, propionic acid, glycolic
acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic
acid, tartaric acid, citric acid, benzoic acid, salicylic acid,
benzenesulphonic acid, and tolunesulphonic acids.
Administration of an "effective amount" of the OX-2 protein and
nucleic acid of the present invention is defined as an amount
effective, at dosages and for periods of time necessary to achieve
the desired result. The effective amount of the OX-2 protein or
nucleic acid of the invention may vary according to factors such as
the disease state, age, sex, and weight of the animal. Dosage
regima may be adjusted to provide the optimum therapeutic response.
For example, several divided doses may be administered daily or the
dose may be proportionally reduced as indicated by the exigencies
of the therapeutic situation.
The inventors have shown that administering OX-2 inhibits the
suppression of growth of tumor cells. Accordingly, the present
invention provides a method of inducing tumor cell growth or
metastasis comprising administering an effective amount of an OX-2
protein or fragment thereof or a nucleic acid sequence encoding an
OX-2 protein or fragment thereof to an animal in need thereof. The
method can be used in experimental systems to study tumor cell
growth or metastasis. The method can also be used to develop an
animal model to study or test chemotherapeutic agents.
The present inventors have shown that there is an association
between levels of OX-2 expression and fertility. In particular the
inventor has shown that low levels (or no levels) of OX-2 is
related to fetal loss. Further, administering a OX-2:Fc fusion
protein prevented fetal loss.
Accordingly, the present invention provides a method of preventing,
reducing or inhibiting fetal loss comprising administering an
effective amount of an OX-2 protein or a nucleic acid sequence
encoding an OX-2 protein to an animal in need thereof. The
invention includes a use of an effective amount of an OX-2 protein
on a nucleic acid molecules encoding an OX-2 protein to prevent or
inhibit fetal loss. The OX-2 protein may be from any species and
may be the full length sequence or a fragment thereof that is
capable of preventing or inhibiting fetal loss. When treating fetal
loss, the animal is a female who is desirous of becoming pregnant
or maintaining a pregnancy.
In another embodiment, the present invention provides a method of
inducing immune tolerance to a transplanted organ or tissue in a
recipient animal comprising administering an effective amount of a
OX-2 protein or a nucleic acid sequence encoding an OX-2 protein to
the recipient animal prior to the transplantation of the organ or
tissue. The invention includes a use of an effective amount of a
OX-2 protein or a nucleic acid sequence encoding an OX-2 protein to
induce immune tolerance to a transplanted organ or tissue.
The term "inducing immune tolerance" means rendering the immune
system unresponsive to a particular antigen without inducing a
prolonged generalized immune deficiency. The term "antigen" means a
substance that is capable of inducing an immune response. In the
case of autoimmune disease, immune tolerance means rendering the
immune system unresponsive to an auto-antigen that the host is
recognizing as foreign, thus causing an autoimmune response. In the
case of allergy, immune tolerance means rendering the immune system
unresponsive to an allergen that generally causes an immune
response in the host. In the case of transplantation, immune
tolerance means rendering the immune system unresponsive to the
antigens on the transplant. An alloantigen refers to an antigen
found only in some members of a species, such as blood group
antigens. A xenoantigen refers to an antigen that is present in
members of one species but not members of another. Correspondingly,
an allograft is a graft between members of the same species and a
xenograft is a graft between members of a different species.
The recipient can be any member of the animal kingdom including
rodents, pigs, cats, dogs, ruminants, non-human primates and
preferably humans. The organ or tissue to be transplanted can be
from the same species as the recipient (allograft) or can be from
another species (xenograft). The tissues or organs can be any
tissue or organ including heart, liver, kidney, lung, pancreas,
pancreatic islets, brain tissue, cornea, bone, intestine, skin and
heamatopoietic cells.
The method of the invention may be used to prevent graft versus
host disease wherein the immune cells in the transplant mount an
immune attack on the recipient's immune system. This can occur when
the tissue to be transplanted contains immune cells such as when
bone marrow or lymphoid tissue is transplanted when treating
leukemias, aplastic anemias and enzyme or immune deficiencies, for
example.
Accordingly, in another embodiment, the present invention provides
a method of preventing or inhibiting graft versus host disease in a
recipient animal receiving an organ or tissue transplant comprising
administering an effective amount of a OX-2 protein or a nucleic
acid sequence encoding an OX-2 protein to the organ or tissue prior
to the transplantation in the recipient animal. The invention
includes a use of an effective amount of an OX-2 protein or a
nucleic acid molecule encoding an OX-2 protein to prevent or
inhibit graft versus host disease.
As stated previously, the method of the present invention may also
be used to treat or prevent autoimmune disease. In an autoimmune
disease, the immune system of the host fails to recognize a
particular antigen as "self" and an immune reaction is mounted
against the host's tissues expressing the antigen. Normally, the
immune system is tolerant to its own host's tissues and
autoimmunity can be thought of as a breakdown in the immune
tolerance system.
Accordingly, in a further embodiment, the present invention
provides a method of preventing or treating an autoimmune disease
comprising administering an effective amount of an OX-2 protein or
a nucleic acid sequence encoding an OX-2 protein to an animal
having, suspected of having, or susceptible to having an autoimmune
disease. The invention includes a use of an effective amount of an
OX-2 protein on a nucleic acid molecule encoding an OX-2 protein to
prevent or inhibit an autoimmune disease.
Autoimmune diseases that may be treated or prevented according to
the present invention include, but are not limited to, type 1
insulin-dependent diabetes mellitus, adult respiratory distress
syndrome, inflammatory bowel disease, dermatitis, meningitis,
thrombotic thrombocytopenic purpura, Sjogren's syndrome,
encephalitis, uveitic, leukocyte adhesion deficiency, rheumatoid
arthritis, rheumatic fever, Reiter's syndrome, psoriatic arthritis,
progressive systemic sclerosis, primary biniary cirrhosis,
pemphigus, pemphigoid, necrotizing vasculitis, myasthenia gravis,
multiple sclerosis, lupus erythematosus, polymyositis, sarcoidosis,
granulomatosis, vasculitis, pernicious anemia, CNS inflammatory
disorder, antigen-antibody complex mediated diseases, autoimmune
haemolytic anemia, Hashimoto's thyroiditis, Graves disease,
habitual spontaneous abortions, Reynard's syndrome,
glomerulonephritis, dermatomyositis, chronic active hepatitis,
celiac disease, autoimmune complications of AIDS, atrophic
gastritis, ankylosing spondylitis and Addison's disease.
As stated previously, the method of the present invention may also
be used to treat or prevent an allergic reaction. In an allergic
reaction, the immune system mounts an attack against a generally
harmless, innocuous antigen or allergen. Allergies that may be
prevented or treated using the methods of the invention include,
but are not limited to, hay fever, asthma, atopic eczema as well as
allergies to poison oak and ivy, house dust mites, bee pollen,
nuts, shellfish, penicillin and numerous others.
Accordingly, in a further embodiment, the present invention
provides a method of preventing or treating an allergy comprising
administering an effective amount of an OX-2 protein or a nucleic
acid sequence encoding an OX-2 protein to an animal having or
suspected of having an allergy. The invention includes a use of an
effective amount of an OX-2 protein or a nucleic acid molecule
encoding an OX-2 protein to prevent or treat an allergy.
Compositions
The invention also includes pharmaceutical compositions containing
OX-2 proteins or nucleic acids for use in immune suppression as
well as pharmaceutical compositions containing an OX-2 inhibitor
for use in preventing immune suppression.
Such pharmaceutical compositions can be for intralesional,
intravenous, topical, rectal, parenteral, local, inhalant or
subcutaneous, intradermal, intramuscular, intrathecal,
transperitoneal, oral, and intracerebral use. The composition can
be in liquid, solid or semisolid form, for example pills, tablets,
creams, gelatin capsules, capsules, suppositories, soft gelatin
capsules, gels, membranes, tubelets, solutions or suspensions.
The pharmaceutical compositions of the invention can be intended
for administration to humans or animals. Dosages to be administered
depend on individual needs, on the desired effect and on the chosen
route of administration.
The pharmaceutical compositions can be prepared by per se known
methods for the preparation of pharmaceutically acceptable
compositions which can be administered to patients, and such that
an effective quantity of the active substance is combined in a
mixture with a pharmaceutically acceptable vehicle. Suitable
vehicles are described, for example, in Remington's Pharmaceutical
Sciences (Remington's Pharmaceutical Sciences, Mack Publishing
Company, Easton, Pa., USA 1985).
On this basis, the pharmaceutical compositions include, albeit not
exclusively, the active compound or substance in association with
one or more pharmaceutically acceptable vehicles or diluents, and
contained in buffered solutions with a suitable pH and iso-osmotic
with the physiological fluids. The pharmaceutical compositions may
additionally contain other agents such as immunosuppressive drugs
or antibodies to enhance immune tolerance or immunostimulatory
agents to enhance the immune response.
In one aspect, the pharmaceutical composition for use in preventing
immune suppression or inhibiting tumor cell growth comprises an
effective amount of a OX-2 inhibitor in admixture with a
pharmaceutically acceptable diluent or carrier. Such compositions
may be administered as a vaccine either alone or in combination
with other active agents.
In one embodiment, the pharmaceutical composition for use in
inhibiting tumor cell growth comprises an effective amount of an
antibody to OX-2 in admixture with a pharmaceutically acceptable
diluent or carrier. The antibodies may be delivered
intravenously.
In another embodiment, the pharmaceutical composition for use in
inhibiting tumor cell growth comprises an effective amount of an
antisense oligonucleotide nucleic acid complimentary to a nucleic
acid sequence from a OX-2 gene in admixture with a pharmaceutically
acceptable diluent or carrier. The oligonucleotide molecules may be
administered as described below for the compositions containing
OX-2 nucleic acid sequences.
When used in inhibiting tumor cell growth or in treating cancer the
composition can additionally contain other agents such as other
immune stimulants (including cytokines and adjuvants) as well as
chemotherapeutic agents.
In another embodiment, the pharmaceutical composition for use in
immune suppression comprises an effective amount of a OX-2 protein
in admixture with a pharmaceutically acceptable diluent or carrier.
The OX-2 protein is preferably prepared as an immunoadhesion
molecule in soluble form which can be administered to the
patient.
In another embodiment, the pharmaceutical composition for use in
immune suppression comprises an effective amount of a nucleic acid
molecule encoding a OX-2 protein in admixture with a
pharmaceutically acceptable diluent or carrier.
The nucleic acid molecules of the invention encoding a OX-2 protein
may be used in gene therapy to induce immune tolerance. Recombinant
molecules comprising a nucleic acid sequence encoding a OX-2
protein, or fragment thereof, may be directly introduced into cells
or tissues in vivo using delivery vehicles such as retroviral
vectors, adenoviral vectors and DNA virus vectors. They may also be
introduced into cells in vivo using physical techniques such as
microinjection and electroporation or chemical methods such as
coprecipitation and incorporation of DNA into liposomes.
Recombinant molecules may also be delivered in the form of an
aerosol or by lavage. The nucleic acid molecules of the invention
may also be applied extracellularly such as by direct injection
into cells. The nucleic acid molecules encoding OX-2 are preferably
prepared as a fusion with a nucleic acid molecule encoding an
immunoglobulin (Ig) Fc region. As such, the OX-2 protein will be
expressed in vivo as a soluble fusion protein.
Murine OX-2
The inventor has cloned and sequenced the murine OX-2 gene.
Accordingly, the invention also includes an isolated nucleic acid
sequence encoding a murine OX-2 gene and having the sequence shown
in FIG. 7 and SEQ. ID. NO.: 1.
The term "isolated" refers to a nucleic acid substantially free of
cellular material or culture medium when produced by recombinant
DNA techniques, or chemical precursors, or other chemicals when
chemically synthesized. The term "nucleic acid" is intended to
include DNA and RNA and can be either double stranded or single
stranded.
Preferably, the purified and isolated nucleic acid molecule of the
invention comprises (a) a nucleic acid sequence as shown in SEQ.
ID. NO.: 1, wherein T can also be U; (b) nucleic acid sequences
complementary to (a); (c) a fragment of (a) or (b) that is at least
15 bases, preferably 20 to 30 bases, and which will hybridize to
(a) or (b) under stringent hybridization conditions; or (a) a
nucleic acid molecule differing from any of the nucleic acids of
(a) or (b) in codon sequences due to the degeneracy of the genetic
code.
It will be appreciated that the invention includes nucleic acid
molecules encoding truncations of the murine OX-2 proteins of the
invention, and analogs and homologs of the proteins of the
invention and truncations thereof, as described below. It will
further be appreciated that variant forms of the nucleic acid
molecules of the invention which arise by alternative splicing of
an mRNA corresponding to a cDNA of the invention are encompassed by
the invention.
An isolated nucleic acid molecule of the invention which is DNA can
also be isolated by selectively amplifying a nucleic acid encoding
a novel protein of the invention using the polymerase chain
reaction (PCR) methods and cDNA or genomic DNA. It is possible to
design synthetic oligonucleotide primers from the nucleic acid
molecules as shown in FIG. 7 for use in PCR. A nucleic acid can be
amplified from cDNA or genomic DNA using these oligonucleotide
primers and standard PCR amplification techniques. The nucleic acid
so amplified can be cloned into an appropriate vector and
characterized by DNA sequence analysis. It will be appreciated that
cDNA may be prepared from mRNA, by isolating total cellular mRNA by
a variety of techniques, for example, by using the
guanidinium-thiocyanate extraction procedure of Chirgwin et al.,
Biochemistry, 18, 5294-5299 (1979). cDNA is then synthesized from
the mRNA using reverse transcriptase (for example, Moloney MLV
reverse transcriptase available from Gibco/BRL, Bethesda, MD, or
AMV reverse transcriptase available from Seikagaku America, Inc.,
St. Petersburg, Fla.).
An isolated nucleic acid molecule of the invention which is RNA can
be isolated by cloning a cDNA encoding a novel protein of the
invention into an appropriate vector which allows for transcription
of the cDNA to produce an RNA molecule which encodes a OX-2 protein
of the invention. For example, a cDNA can be cloned downstream of a
bacteriophage promoter, (e.g. a T7 promoter) in a vector, cDNA can
be transcribed in vitro with T7 polymerase, and the resultant RNA
can be isolated by standard techniques.
A nucleic acid molecule of the invention may also be chemically
synthesized using standard techniques. Various methods of
chemically synthesizing polydeoxynucleotides are known, including
solid-phase synthesis which, like peptide synthesis, has been fully
automated in commercially available DNA synthesizers (See e.g.,
Itakura et al. U.S. Pat. No. 4,598,049; Caruthers et al. U.S. Pat.
No. 4,458,066; and Itakura U.S. Pat. Nos. 4,401,796 and
4,373,071).
The sequence of a nucleic acid molecule of the invention may be
inverted relative to its normal presentation for transcription to
produce an antisense nucleic acid molecule. Preferably, an
antisense sequence is constructed by inverting a region preceding
the initiation codon or an unconserved region. In particular, the
nucleic acid sequences contained in the nucleic acid molecules of
the invention or a fragment thereof, preferably a nucleic acid
sequence shown in FIG. 7 may be inverted relative to its normal
presentation for transcription to produce antisense nucleic acid
molecules.
The antisense nucleic acid molecules of the invention or a fragment
thereof, may be chemically synthesized using naturally occurring
nucleotides or variously modified nucleotides designed to increase
the biological stability of the molecules or to increase the
physical stability of the duplex formed with mRNA or the native
gene e.g. phosphorothioate derivatives and acridine substituted
nucleotides. The antisense sequences may be produced biologically
using an expression vector introduced into cells in the form of a
recombinant plasmid, phagemid or attenuated virus in which
antisense sequences are produced under the control of a high
efficiency regulatory region, the activity of which may be
determined by the cell type into which the vector is
introduced.
The invention also provides nucleic acids encoding fusion proteins
comprising a OX-2 protein of the invention and a selected protein,
or a selectable marker protein.
The invention further includes an isolated protein which has the
amino acid sequence as shown in FIG. 8 and SEQ. ID. NO.: 2.
Within the context of the present invention, a protein of the
invention may include various structural forms of the primary
protein which retain biological activity. For example, a protein of
the invention may be in the form of acidic or basic salts or in
neutral form. In addition, individual amino acid residues may be
modified by oxidation or reduction.
In addition to the full length amino acid sequence (FIG. 8), the
protein of the present invention may also include truncations of
the protein, and analogs, and homologs of the protein and
truncations thereof as described herein. Truncated proteins may
comprise peptides of at least fifteen amino acid residues.
Analogs of the protein having the amino acid sequence shown in FIG.
8, and/or truncations thereof as described herein, may include, but
are not limited to an amino acid sequence containing one or more
amino acid substitutions, insertions, and/or deletions. Amino acid
substitutions may be of a conserved or non-conserved nature.
Conserved amino acid substitutions involve replacing one or more
amino acids of the proteins of the invention with amino acids of
similar charge, size, and/or hydrophobicity characteristics. When
only conserved substitutions are made the resulting analog should
be functionally equivalent. Non-conserved substitutions involve
replacing one or more amino acids of the amino acid sequence with
one or more amino acids which possess dissimilar charge, size,
and/or hydrophobicity characteristics.
One or more amino acid insertions may be introduced into the amino
acid sequences shown in FIG. 8. Amino acid insertions may consist
of single amino acid residues or sequential amino acids ranging
from 2 to 15 amino acids in length. For example, amino acid
insertions may be used to render the protein is no longer active.
This procedure may be used in vivo to inhibit the activity of a
protein of the invention.
Deletions may consist of the removal of one or more amino acids, or
discrete portions from the amino acid sequence shown in FIG. 8. The
deleted amino acids may or may not be contiguous. The lower limit
length of the resulting analog with a deletion mutation is about 10
amino acids, preferably 100 amino acids.
Analogs of a protein of the invention may be prepared by
introducing mutations in the nucleotide sequence encoding the
protein. Mutations in nucleotide sequences constructed for
expression of analogs of a protein of the invention must preserve
the reading frame of the coding sequences. Furthermore, the
mutations will preferably not create complementary regions that
could hybridize to produce secondary mRNA structures, such as loops
or hairpins, which could adversely affect translation of the
receptor mRNA.
Mutations may be introduced at particular loci by synthesizing
oligonucleotides containing a mutant sequence, flanked by
restriction sites enabling ligation to fragments of the native
sequence. Following ligation, the resulting reconstructed sequence
encodes an analog having the desired amino acid insertion,
substitution, or deletion.
Alternatively, oligonucleotide-directed site specific mutagenesis
procedures may be employed to provide an altered gene having
particular codons altered according to the substitution, deletion,
or insertion required. Deletion or truncation of a protein of the
invention may also be constructed by utilizing convenient
restriction endonuclease sites adjacent to the desired deletion.
Subsequent to restriction, overhangs may be filled in, and the DNA
religated. Exemplary methods of making the alterations set forth
above are disclosed by Sambrook et al (Molecular Cloning: A
Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press,
1989).
The invention also contemplates isoforms of the proteins of the
invention. An isoform contains the same number and kinds of amino
acids as a protein of the invention, but the isoform has a
different molecular structure. The isoforms contemplated by the
present invention are those having the same properties as a protein
of the invention as described herein.
The present invention also includes a protein of the invention
conjugated with a selected protein, or a selectable marker protein
to produce fusion proteins. Additionally, immunogenic portions of a
protein of the invention are within the scope of the invention.
The proteins of the invention (including truncations, analogs,
etc.) may be prepared using recombinant DNA methods. Accordingly,
the nucleic acid molecules of the present invention having a
sequence which encodes a protein of the invention may be
incorporated in a known manner into an appropriate expression
vector which ensures good expression of the protein. Possible
expression vectors include but are not limited to cosmids,
plasmids, or modified viruses (e.g. replication defective
retroviruses, adenoviruses and adeno-associated viruses), so long
as the vector is compatible with the host cell used. The expression
vectors are "suitable for transformation of a host cell", means
that the expression vectors contain a nucleic acid molecule of the
invention and regulatory sequences selected on the basis of the
host cells to be used for expression, which is operatively linked
to the nucleic acid molecule. Operatively linked is intended to
mean that the nucleic acid is linked to regulatory sequences in a
manner which allows expression of the nucleic acid.
The invention therefore contemplates a recombinant expression
vector of the invention containing a nucleic acid molecule of the
invention, or a fragment thereof, and the necessary regulatory
sequences for the transcription and translation of the inserted
protein-sequence. Such expression vectors may be useful in the
above-described therapies using a nucleic acid sequence encoding a
OX-2 protein. Suitable regulatory sequences may be derived from a
variety of sources, including bacterial, fungal, or viral genes
(For example, see the regulatory sequences described in Goeddel,
Gene Expression Technology: Methods in Enzymology 185, Academic
Press, San Diego, Calif. (1990). Selection of appropriate
regulatory sequences is dependent on the host cell chosen, and may
be readily accomplished by one of ordinary skill in the art.
Examples of such regulatory sequences include: a transcriptional
promoter and enhancer or RNA polymerase binding sequence, a
ribosomal binding sequence, including a translation initiation
signal. Additionally, depending on the host cell chosen and the
vector employed, other sequences, such as an origin of replication,
additional DNA restriction sites, enhancers, and sequences
conferring inducibility of transcription may be incorporated into
the expression vector. It will also be appreciated that the
necessary regulatory sequences may be supplied by the native
protein and/or its flanking regions.
The invention further provides a recombinant expression vector
comprising a DNA nucleic acid molecule of the invention cloned into
the expression vector in an antisense orientation. That is, the DNA
molecule is operatively linked to a regulatory sequence in a manner
which allows for expression, by transcription of the DNA molecule,
of an RNA molecule which is antisense to a nucleotide sequence
comprising the nucleotides as shown in FIG. 7. Regulatory sequences
operatively linked to the antisense nucleic acid can be chosen
which direct the continuous expression of the antisense RNA
molecule.
The recombinant expression vectors of the invention may also
contain a selectable marker gene which facilitates the selection of
host cells transformed or transfected with a recombinant molecule
of the invention. Examples of selectable marker genes are genes
encoding a protein such as G418 and hygromycin which confer
resistance to certain drugs, .beta.-galactosidase, chloramphenicol
acetyltransferase, or firefly luciferase. Transcription of the
selectable marker gene is monitored by changes in the concentration
of the selectable marker protein such as b-galactosidase,
chloramphenicol acetyltransferase, or firefly luciferase. If the
selectable marker gene encodes a protein conferring antibiotic
resistance such as neomycin resistance transformant cells can be
selected with G418. Cells that have incorporated the selectable
marker gene will survive, while the other cells die. This makes it
possible to visualize and assay for expression of recombinant
expression vectors of the invention and in particular to determine
the effect of a mutation on expression and phenotype. It will be
appreciated that selectable markers can be introduced on a separate
vector from the nucleic acid of interest.
The recombinant expression vectors may also contain genes which
encode a fusion moiety which provides increased expression of the
recombinant protein; increased solubility of the recombinant
protein; and aid in the purification of a target recombinant
protein by acting as a ligand in affinity purification. For
example, a proteolytic cleavage site may be added to the target
recombinant protein to allow separation of the recombinant protein
from the fusion moiety subsequent to purification of the fusion
protein.
Recombinant expression vectors can be introduced into host cells to
produce a transformant host cell. The term "transformant host cell"
is intended to include prokaryotic and eukaryotic cells which have
been transformed or transfected with a recombinant expression
vector of the invention. The terms "transformed with", "transfected
with", "transformation" and "transfection" are intended to
encompass introduction of nucleic acid (e.g. a vector) into a cell
by one of many possible techniques known in the art. Prokaryotic
cells can be transformed with nucleic acid by, for example,
electroporation or calcium-chloride mediated transformation.
Nucleic acid can be introduced into mammalian cells via
conventional techniques such as calcium phosphate or calcium
chloride co-precipitation, DEAE-dextran-mediated transfection,
lipofectin, electroporation or microinjection. Suitable methods for
transforming and transfecting host cells can be found in Sambrook
et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold
Spring Harbor Laboratory press (1989)), and other laboratory
textbooks.
Suitable host cells include a wide variety of prokaryotic and
eukaryotic host cells. For example, the proteins of the invention
may be expressed in bacterial cells such as E. coli, insect cells
(using baculovirus), yeast cells or mammalian cells. Other suitable
host cells can be found in Goeddel, Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1991).
The proteins of the invention may also be prepared by chemical
synthesis using techniques well known in the chemistry of proteins
such as solid phase synthesis (Merrifield, 1964, J. Am. Chem.
Assoc. 85:2149-2154) or synthesis in homogenous solution
(Houbenweyl, 1987, Methods of Organic Chemistry, ed. E. Wansch,
Vol. 15 I and II, Thieme, Stuttgart).
The following non-limiting examples are illustrative of the present
invention:
EXAMPLES
Example 1
This example demonstrates the increased expression of certain genes
following pv immunization.
Mice:
C3H/HeJ and C57BL/6 mice were purchased from The Jackson
Laboratory, Bar Harbor, Me. Mice were housed five/cage and allowed
food and water ad libitum. All mice were used at 8-12 weeks of
age.
Monoclonal Antibodies:
The following monoclonal antibodies (Mabs) from Pharmingen (San
Diego, Calif.) were used: anti-IL-2 (JES6-1A12; biotinylated
JES6-5H4); anti-IL-4 (11B 11; biotinylated BVD6-24G2);
anti-IFN.gamma. (R4-6A2; biotinylated XMG1.2); anti-IL-10
(JES5-2A5; biotinylated SXC-1, Pharmingen, San Diego, Calif.);
mouse IgG1 isotype control (clone 107.3, BALB/c anti-TNP).
Strepavidin horse radish peroxidase and recombinant mouse GM-CSF
was also purchased from Pharmingen (San Diego, Calif.).
NLDC-145 (anti-mouse dendritic cells), and F(ab').sub.2 rabbit
anti-rat IgG FITC conjugate (non-cross reactive with mouse IgG), or
F(ab').sub.2 rabbit anti-mouse IgG PE was obtained from Serotec,
Canada.
Rabbit complement, L3T4, anti-thy1.2, anti-Ly2.2, anti-Ly2.1 (mouse
IgG3), FITC-MAC-1 and mouse IgG1 anti-rat OX-2 were obtained from
Cedarlane Labs, Hornby, Ontario.
Anti-CD28 (PV-1) and anti-CTLA (UC10-4F10-11) were obtained from
Drs. C. June and J. Bluestone respectively, while anti-B7-1,
anti-B7-2 were obtained from Dr. G. Powers. High titres of all 4 of
the latter antibodies were produced by in vitro culture in a
CELLMAX system (CELLCO Inc., Germantown, Md., USA).
Preparation of Cells:
Spleen, Peyer's Patch (PP) and mesenteric lymph node (MLN) cell
suspensions were prepared aseptically from individual mice of the
different treated groups in each experiment.
Where dendritic cells were obtained by culture of bone marrow cells
in vitro the following technique was used (Gorczynski et al.,
1996a). Bone marrow plugs were aspirated from the femurs of donor
male C57BL/6 (or BALB/c) mice, washed and resuspended in aF10.
Cells were treated sequentially with a mixture of antibodies (L3T4,
anti-thy1.2, anti-Ly2.2) and rabbit complement and dead cells
removed by centrifugation over mouse lymphopaque (Cedarlane Labs,
Ontario). Cells were washed x3 in aF10, and cultured in 10 ml aF10
in tissue culture flasks, at a concentration of 2.times.10.sup.6
/ml with 500 U/ml recombinant murine GM-CSF (Pharmingen, USA).
Fresh GM-CSF was added at 36 hr intervals. Cells were separated
over lymphopaque on days 3.5 and 7 of culture, again reculturing in
aF10 with recombinant GM-CSF. At 10 days an aliquot of the sample
was stained with NLDC-145 and FITC anti-rat IgG, anti-OX-2 and PE
antimouse IgG, FITC-anti-B7-1 or FITC anti-B7-2. Mean staining with
these antibodies using cells harvested from such cultures has been
93%.+-.7%, 14%.+-.5%, 78%.+-.9% and 27%.+-.6% respectively.
Remaining cells were washed, and injected into the portal vein as
described.
Portal Vein Immunizations and Renal Transplantation:
The pv immunizations and renal transplantation were performed as
described earlier (Gorczynski et al., 1994). All C3H mice received
pv/iv immunization with 15.times.10.sup.6 C57BL/6 10-day cultured,
bone marrow derived, dendritic cells, followed by C57BL/6 kidney
transplantation. Animals received 1 intramoscular (im) injection
with 10 mg/Kg cyclosporin A on the day of transplantation. Mice
were sacrificed for tissue harvest and RNA preparation 5 days after
transplantation. In other studies animals were sacrificed as
described in the text.
Where monoclonal antibodies were injected into transplanted mice,
animals received 100 mg intravenous (iv) at 2 day intervals
(.times.4 injections) beginning within 2 hours of
transplantation.
Cytokine Production from Spleen Cells of Transplanted Mice:
In cultures used to assess induction of cytokine production spleen
responder cells stimulated with irradiated (2000R) C57BL/6 spleen
stimulator cells in triplicate in aF10 have been used. In multiple
studies significant quantitative or qualitative differences in
cytokine production from spleen, lymph node or Peyer's Patch of
transplanted mice have not been seen. (Gorczynski et al., 1994b).
Supernatants were pooled at 40 hr from replicate wells and assayed
in triplicate in ELISA assays for lymphokine production. All
capture antibodies, biotinylated detection antibodies, and
recombinant cytokines were obtained from Pharmingen (San Diego,
Calif.--see above).
For IFN.gamma. the assay used flat-bottomed 96-well Nunc plates
(Gibco, BRL) coated with 100 ng/ml R4-6A2. Varying volumes of
supernatant were bound in triplicate at 4.degree. C., washed x3,
and biotinylated anti-IFN.gamma. (XMG1.2) added. After washing,
plates were incubated with strepavidin-horse radish peroxidase
(Cedarlane Labs), developed with appropriate substrate and
OD.sub.405 determined using an ELISA plate reader. IL-10 was
assayed using a similar ELISA system with JES5-2A5 as the capture
antibody, and biotinylated SXC-1 as developing antibody. Each assay
reliably detected cytokine levels in the range 0.01 to 0.1 ng/ml.
ELISA assays for IL-2 and IL-4 used JES6-1A12 and 11B11 as capture
antibodies, with biotinylated JES6-5H4 or BVD6-24G2 as developing
antibodies. Sensitivity of detection was 10 pg/ml for each
cytokine.
Oligonucleotide Primers:
The primers used for PCR amplification for b-actin, and different
cytokines, are described in previous publications (Gorczynski, R.
M., 1995a; Gorczynski, R. M., 1995b; Gorczynski, R. M., 1996a). In
addition, the following oligonucleotides were synthesized.
cDNA synthesis primer for driver ds cDNA (DP):
(SEQ.ID.NO.:3) 5'-TTTTGTACAAGCTT.sub.30 -3' Adapter 1 (Ad1):
(SEQ.ID.NO.:4) 5'-CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT-3'
Adapter 2 (Ad2): (SEQ.ID.NO.:5)
5'-TGTAGCGTGAAGACGACAGAAAGGGCGTGGTGCGGAGGGCGGT-3' PCR Primer1 (P1):
(SEQ.ID.NO.:6) 5'-CTAATACGACTCACTATAGGGC-3' Nested Primer 1 (NP1):
(SEQ.ID.NO.:7) 5'-TCGAGCGGCCGCCCGGGCAGGT-3' PCR Primer2 (P2):
(SEQ.ID.NO.:8) 5'-TGTAGCGTGAAGACGACAGAA-3' Nested Primer 2 (NP2):
(SEQ.ID.NO.:9) 5'-AGGGCGTGGTGCGGAGGGCGGT-3'
Driver and Tester Preparation:
RNA was extracted from pooled mesenteric lymph node (MLN) and
Peyer's Patches (PP) of 5/group renal transplant mice with iv or pv
immunization. Poly(A).sup.+ mRNA was prepared from the driver (iv)
group, and 2 mg material used for ds cDNA synthesis with 1 ng DP
primer and a cDNA Synthesis Kit (Clontech) with T4 DNA polymerase.
The final cDNA preparation was digested with Rsal in a 50 ml
reaction mixture with 15 units enzyme (GIBCO) for 3 hrs, and the
cDNA phenol-extracted, ethanol precipitated, and resuspended in 7
ml of deionized water (concentration approximately 300 ng/ml).
Rsal digested ds tester cDNA (pv group) was prepared in a similar
fashion. 50 ng of tester CDNA diluted in TE buffer was ligated with
2 ml of Ad1 and Ad2 (each at 10 mM) in separate ligation reactions
at 16.degree. C. for 18 hrs with 50 Units/ml T4 ligase. Thereafter
1 ml of 0.2M EDTA was added, the mixture heated at 70.degree. C.
for 5 min to inactivate the ligase, and the product stored at
-70.degree. C.
Subtractive Hybridization and PCR Amplification:
600 ng driver (iv) ds cDNA was added to each of two tubes
containing 20 ng Ad1- and Ad2-ligated pv cDNA. The samples were
mixed, precipitated with ethanol, resuspended in hybridization
buffer, overlaid with mineral oil and denatured/annealed in
standard fashion. The two independent samples were then combined,
200 ng fresh driver cDNA added to allow for further enrichment of
differentially expressed mRNAs, and the mixture again denatured and
annealed for 10 hrs at 68.degree. C. The final sample was diluted
in Hepes buffer with EDTA and stored at -20.degree. C.
After subtraction two PCR amplifications were performed on the
subtracted cDNA. In the first 1 ml of subtracted cDNA was amplified
using 1 ml each of P1 and P2. The conditions for amplification were
as described by Diatchenko. The amplified products were diluted
10-fold in deionized water and 1 ml of product used for further
amplification using the nested primers (NP1 and NP2) and a 10-cycle
amplification reaction. Aliquots of the original driver/tester and
subtracted cDNAs were used for PCR reactions with control
oligonucleotide primers (b-actin) for known "housekeeping genes",
and with primers for genes whose expression has been previously
documented to be different in iv/pv immunized mice. These data are
shown in FIGS. 1 and 2.
FIG. 1 shows PCR validation of suppressive subtractive
hybridization. Samples from unsubtracted (lanes 1, 3, 5 and 7) or
subtracted (lanes 2, 4, 6 and 8) mRNA were reverse transcribed and
tested in PCR with b-actin primers for different PCR cycle times.
Lanes 1 and 2: 15 cycles; lanes 3 and 4: 20 cycles; lanes 5 and 6:
25 cycles; lanes 7 and 8: 30 cycles.
FIG. 2 shows PCR validation of suppressive subtractive
hybridization. Samples from unsubtracted (lanes 2 and 4) or
subtracted (lanes 3 and 5) mRNA were tested as in FIG. 1, except
primers used were for IL-10, and different cycle times are shown.
Lanes 2 and 3: 20 cycles; lanes 4 and 5: 30 cycles, lane 1: mol.
wt. standard.
In addition, cloning of the subtracted cDNA was performed as
follows.
Cloning and Further Analysis of Subtracted cDNA:
The PCR amplified cDNA was cloned with a TA cloning kit
(Invitrogen, Calif.) by directly ligating into the PCR II vector.
Ligation was performed at an insert:vector ratio of 3:1 in 1.times.
ligation buffer with T4 ligase (3 U/ml) overnight at 14.degree. C.
Ligation products were then inserted into INFaF' competent
Escherichia Coli using a standard transformation protocol, and
selected with ampicillin on plates containing X-gal
(5-bromo-4-chloro-3-indolyl-D-galactoside). Miniprep plasmid DNA
was purified with a Plasmid extraction Spin kit (Qiagen, Germany)
and cut with EcoR I restriction enzyme to determine whether the
plasmids contained the expected insert. Plasmids with inserts were
sequenced by the dideoxy sequencing method using a T7 sequencing
kit (Pharmacia Biotech, Canada). Nucleic acid homology searches
were performed using the BLAST program at the National Center for
Biotechnology Information (NIH, Bethesda, USA).
Further analyses of cloned material, using Northern hybridization,
was as follows. Inserts in pCRII were amplified for 12 cycles using
nested PCR primers. The amplified material was purified using
Qiaquick Spin PCR Purification Kits (Qiagen), .sup.32 P-labeled by
random priming, and used as a probe for Northern hybridization with
20 mg samples of the original (and fresh) iv or pv total RNA.
Hybridization was performed in 5 ml of ExpressHyb solution
(Clontech) with a minimum of 5.times.10.sup.6 cpm per 10 ng cDNA
probe and 0.1 mg/ml sonicated heat-denatured salmon sperm DNA.
Filters were washed 4 times, each at 15 min at 27.degree. C. with
1.times.SSC and 0.1% SDS, followed by a high stringency wash at
42.degree. C. for 30 min with 0.2.times.SSC and 0.1% SDS. Exposure
times varied from 18 hrs to 6 days. FIG. 3 shows an autoradiograph
using .sup.32g P-labeled probes prepared from 4 clones obtained
using the subtraction hybridization approach described above (with
pv cDNA as tester material and iv cDNA as driver). A labeled
control probe was prepared with a PCR amplicon for mouse b-actin.
Total RNA was prepared from mice receiving iv or pv immunization
and equivalent amounts loaded in replicate lanes as shown, with
gels developed from 18 hours (clone #28) to 6 days (#71). Clone 8
is most homologous with mouse poly (A) binding protein. Clone 16 is
most homologous with rat MRC OX-2. Clone 28 is most homologous with
human zinc-finger protein. Clone 71 has no homologous sequence.
Western Blotting Protocol:
The technique used was essentially that described by Sandhu et al.
(1991) as modified by Bronstein et al. (1992). Samples were
obtained 14 days post renal transplantation, using groups described
in FIG. 5. Fresh rat thymus cells were used as control. Samples
were electrophoresed in 12% SDS-PAGE and transferred to PVDF
membranes (Novex Co., San Diego, Calif.) prior to addition of
primary antibody. A commercial anti-rat OX-2 was used as test
reagent; control antibody was an antibody to mouse CD8a. The
developing antibody used was a commercial horse-radish peroxidase
labeled anti-mouse IgG. All reagents were obtained from Cedarlane
Labs (Hornby, Ontario, Canada).
DNA Sequence Homology Comparison:
Comparison of mouse OX-2 with known cDNA sequences for B7-1, B7-2,
CD28 and CTLA-4 was performed using a DNASIS program (version
2.0).
Results
Evaluation of Suppression Subtraction Hybridization(SSH)
Technique
In order to evaluate the efficacy of the SSH technique used, the
inventor used his previous evidence that, by PCR analysis,
increased expression of mRNA for IL-10 genes was evident in
lymphoid tissue from pv immunized mice. Accordingly, a dilution
analysis of cDNA from the tester, driver and subtracted material,
using PCR primers for b-actin and IL-10 was performed. As shown in
FIG. 1, after SSH there was a detectable signal for b-actin in
subtracted material only after 35 cycles of amplification. By
contrast, a signal was present in the unsubtracted material after
only 15 cycles. Using additional quantitative measures of template,
it was found to correspond to some 1000-10,000 depletion of b-actin
mRNA. In a separate study, analyzing IL-10 mRNA (FIG. 2),
significant enrichment of IL-10 mRNA was found as determined by
comparison of the amplification detected at 30 cycles in
subtracted/unsubtracted material (see lanes 4 and 5, FIG. 2).
In a further test of the efficiency of subtraction the mixture of
unsubtracted and subtracted tester (pv) cDNA was labeled and
hybridized to Northern blots of iv (tester) and pv (driver) total
RNA. The results (data not shown) indicated that the subtracted
tester cDNA probe did indeed produce a significantly stronger
signal with the tester RNA. Given the evidence that for any cDNA
species to produce a signal in a Norther blot it must represent a
concentration greater than 0.1-0.3% of the cDNA mixture, these data
are again consistent with our having produced a high level of
enrichment of pv-specific cDNA, with a concomitant reduction in
abundant cDNAs common between tester (pv) and driver (iv)
material.
Detection of Unique cDNA Fraqments in Tissue from pv Immunized
Mice
The efficiency and validity of SSH for detection of cDNAs unique to
the tissue sample from the pv immunized mice was further confirmed
after cloning and sequence analysis of selected tester-specific
cDNAs. 10 randomly selected cDNA clones (of 66 sequenced) were used
to probe multiple preparations of pv or iv whole RNA. All revealed
unique mRNAs expressed preferentially in the pv samples.
Autoradiograms from 4 of these Northern blots, along with a b-actin
probe as control, are shown in FIG. 3. Exposure times from 18 hrs
to 6 days were used which were interpreted as indicative of pv
specific cDNAs of different abundance in the samples of
interest.
The cDNA inserts of the 4 clones shown, along with the other 62
clones, were partially sequenced and analyzed for homology in the
GenBank and EMBL data bases. A summary of these data are shown in
Table 1. Note that some 30 cDNA fragments had at least 50% homology
(BLAST score >250 over at least 50 nt) with other described
sequences. A further 14 clones showed similar homology with known
rat/human genes. Both sets may represent members of different gene
families. An additional 22 clones demonstrated no significant
matches with entries in the database, and thus may represent novel
genes up-regulated after pv immunization. That the data shown are a
minimal estimate of such differentially expressed genes is evident
from the fact that homology with IL-4 or IL-10 gene sequences
(mRNAs known to be over-expressed following pv immunization--see
also FIG. 2) were NOT detected in any of the 66 clones
analyzed.
The sequence homology for the clones shown in FIG. 3 (>80%
homology over the compared sequence) led to the further
characterization of these clones. Clone 8 was shown to be most
homologous with mouse poly (A) binding protein; clone 16 was shown
to be most homologous with rat MRC OX-2; and clone 28 was shown to
be most homolgous with human zinc-finger protein. No homologous
sequence was found for clone 71. In the data that follows, the
analysis of one of these clones which showed homology to a rat cDNA
(for OX-2, a molecule previously characterized as being
preferentially expressed on rat thymocytes and dendritic cells) is
described. The rationale for further investigation of this clone
lies in data showing that infusion of dendritic cells via the
portal vein is a potent method for prolonging allograft survival in
our model systems. Note, however, that while the bone marrow
derived dendritic cells that were infused via the portal vein
themselves express OX-2 (see above), identical data has been
obtained in Northern gels to those shown in FIG. 3 using tissue
harvested from mice receiving, as the earliest studies(1-5)
irradiated spleen cells (OX-2.sup.- by FACS analysis) via the
portal vein. In addition, in both situations, OX-2 mRNA was not
detected by this suppression subtraction hybridization approach
when we used tissue harvested at 0.5-2.5 days post transplantation.
These results are consistent with the idea that the OX-2 signal
detected is a result of novel increased expression in cells
following pv immunization.
Probing a cDNA Library from Tissue from pv Immunized Mice for
Expression of the Murine Equivalent of Rat OX-2
A cDNA library was constructed from mRNA prepared from a pool of 5
C3H mice receiving pv immunization with 25.times.10.sup.6
irradiated (2000 Rads) C57BL/6 bone marrow cells followed by renal
transplantation as described in the Materials and Methods, using a
kit purchased from ClonTech. Clones were plated in LB medium and
probed with the .sup.32g P-labeled amplicon described in FIG. 3 as
showing homology with rat OX-2. A 1.3 Kb clone was detected,
amplified, and shown after .sup.32 P labeling to detect a
differentially expressed product by Northern gel analysis. After
sequencing using an automated DNA sequencer and fluorescent-labeled
deoxynucleotides, this 1.3 Kb fragment was found to share >95%
homology with the region encoding the 3'untranslated region of the
rat OX-2 mRNA as determined from the GeneBank sequence for rat
OX-2.
Using a primer construct program, a 5'PCR primer representing
positions 1-19 of the rat GeneBank sequence (corresponding to a
portion of the 5'untranslated region, and the leader sequence) and
3' primers from our characterized mouse sequence were synthesized,
and long-distance amplification performed to produce an amplicon
predicted to encode the open-reading-frame (ORF) of the murine
equivalent of the rat OX-2 gene. This amplicon was determined (as
expected) to be of some 1.4 Kb length. Automated sequencing
produced a full-length sequence for the mouse homologue of the rat
MRC OX-2 gene, including an ORF with >90% homology (predicted
amino acid sequence) with the corresponding rat product, along with
the 3'untranslated region. This sequence has been submitted to the
Genebank (accession number AF004023).
Using a DNASIS program the predicted mouse protein sequence has
some 51% homology with B7-1 and B7-2, 48% with CD28 and 54% with
CTLA4 (unpublished).
Evidence for an Important Role for the Expressed OX-2 Homologue in
Prolonged Graft Survival Following pv Immunization
In an attempt to define the potential importance of the product
encoded by the OX-2 gene we used a commercial antibody to rat OX-2
in a transplant model in mice receiving pv immunization and renal
transplantation. In the first such study, it was asked whether
there was evidence for specifically increased expression of the
OX-2 molecule following pv immunization. By FACS analysis, using
dual staining of hepatic mononuclear cells and spleen cells with
OX-2 and NLDC145, similar numbers of NLDC145.sup.+ cells in liver
or spleen samples from iv and pv immunized mice were found,
(5.times.10.sup.5 and 6.5.times.10.sup.6 respectively), but a
4-fold increase in the numbers of OX-2.sup.+ NLDC145.sup.+
following pv immunization. FIG. 4 shows a flow cytometry profile of
spleen adherent cells from iv immunized/grafted mice (panels A and
B) or pv immunized/grafted mice (panels C and D). Cells were
harvested 7 days after transplantation and stained with NLDC145 and
F(ab').sub.2 FITC-anti-rat IgG, as well as with control (clone
107.3) mouse IgG1 serum (left hand panels) or anti-OX-2 (right hand
panels) and F(ab').sub.2 PE-anti-mouse IgG. Data are representative
of one of three different studies. Values shown represent the total
cell population in each quadrant. The absolute numbers
(.times.10.sup.5) of double positive cells in the liver or spleen
of pv immunized mice were 3.2.+-.0.5 and 39.+-.8 respectively (see
FIG. 4 for FACS profiles of spleen adherent cells). This 4-fold
increase was seen regardless of the cells used for pv immunization,
either bone marrow derived dendritic cells (some 20% OX-2.sup.+
--see above) or irradiated whole spleen lymphoid cells
(OX-2.sup.-), suggesting that they were not merely detecting
surviving OX-2.sup.+ (donor) cells, but novel expression of OX-2 in
vivo.
Western blot, FIG. 5, shows increased expression of OX-2 antigen
after pv immunization. The technique used for Western blotting is
previously described. Samples were obtained 14 days post renal
transplantation, using the groups described in FIG. 6. Fresh rat
thymus cells (lane 5) were used as control. Lanes 1 and 2 represent
samples pooled from 3 donors/group (iv immunized; pv
immunized+infusion of anti-OX-2 respectively). Samples in lanes 3
and 4 are from individual mice receiving pv immunization and renal
transplantation only (no antibody treatment). Staining with
anti-rat MRC OX-2 is shown in FIG. 5B; with a control antibody (to
mouse Ly2.1), anti-mouse cD8a, shown in FIG. 5A. The developing
antibody used was a commercial horse-radish peroxidase labeled
anti-mouse IgG. No signal was seen using the mouse IgG1 isotype
control clone 107.3 (BALB/c anti-TNP)-data not shown. Data are
representative of 1 of 3 equivalent studies.
Western blotting (see FIGS. 5A and 5B) of samples prepared from the
spleen of iv vs pv immunized and grafted mice 14 days following
renal transplantation revealed staining of a band migrating with
estimated molecular weight 43 Kd, in agreement with data elsewhere
reporting extensive glycosylation of this molecule in isolates from
rat thymus. In mice receiving pv immunization along with in vivo
treatment with anti-OX-2, no detectable signal was seen in Western
blots (see lane 2, FIG. 5). No staining was seen with a murine IgG1
isotype control (BALB/c anti-TNP, clone 107.3: unpublished), making
it unlikely that the band observed was Fc receptor.
FIG. 6 is a graft showing percent survival versus days post renal
transplantation.Commercial anti-OX-2 monoclonal antibody, but not
anti-mouse CD28 or anti-mouse CTLA4, reverses the graft
prolongation following donor-specific pv immunization. Groups of 6
C3H mice received C57BL/6 renal allografts with no other treatment
(cyclosporin A only, -.diamond.-), or additional pv immunization
with 15.times.10.sup.6 C57BL/6 bone marrow derived dendritic cells
(-<-)as described previously. Subsets of these latter mice
received iv injection (every second day .times.4 injections) with
100 mg/mouse of a commercial anti-rat OX-2 monoclonal antibody (--)
or the isotype control (clone 107.3, -), or of antibodies to mouse
CD28 (-.cndot.-) or CTLA4 (-*-) The animal survival for the
different groups shown are pooled from 2 studies. Note that the
mouse isotype control itself produced no modification of the
increased renal graft survival following pv immunization.
*p<0.02, Mann-Whitney U-test).
In two final studies mice received pv immunization and
transplantation as before, but now also received iv injection with
commercial anti-rat OX-2 (.times.4 injections; 100 mg/mouse at 2
day intervals). As shown in FIGS. 5A and B and 6 these infusions of
anti-OX-2 significantly decreased the prolonged graft survival
(FIG. 6) and increased expression of OX-2 antigen (Western
blotting--FIGS. 5A and 5B) seen following pv immunization. No
perturbation of graft survival following pv immunization was seen
using additional treatments with anti-CD28/anti-CTLA4 (see FIG. 6),
or, in studies not shown, using anti-B7-1 or anti-B7-2. Again
infusion of the IgG1 isotype control Mab (clone 107.3) did not
alter the increased graft survival seen following pv immunization
(see FIG. 6).
In separate experiments cells were harvested from mice receiving pv
immunization along with additional treatment with monoclonal
antibodies as show (see Table 2). Following treatment with
anti-OX-2 there was no longer the altered cytokine production (with
polarization to production of IL-4 and IL-10) which the inventor
has described in multiple model systems in which animals received
pv donor-specific pre-transplant immunization. Treatment with any
of the other 4 monoclonal antibodies tested did not produce this
reversal in polarization of cytokine production seen following pv
immunization-indeed, using these Mabs alone in the absence of pv
immunization produced a trend to increased graft survival (not
shown) and significant polarization in cytokine production to
increased IL-4 and IL-10 production, akin to that produced by pv
immunization itself (upper half of Table 2).
OX-2 is a molecule previously characterized by Barclay et al.
(1981, 1982) as being preferentially expressed on rat thymocytes
and dendritic cells. Dendritic cells are known to be important
signalling cells for lymphocytes, which also potentially regulate
cytokine production and graft rejection, and infusion of dendritic
cells is a potent means of inducing pv tolerance. The inventor has
determined that OX-2 expression increased following pv
immunization, and further studied whether this had any functional
consequences. As shown in FIGS. 4 and 5, there is indeed
significantly increased expression of OX-2 in spleen cells isolated
from pv immunized mice, along with the increased graft survival and
polarization in cytokine production (FIG. 6 and Table 2). In
contrast, in vivo infusion of anti-OX-2 abolishes increased
expression of this molecule, simultaneously reversing the increased
graft survival and altered cytokine profile seen. This data is
consistent with the possible function of OX-2.sup.+ cells in
promoting allograft survival.
In the studies described the donor dendritic cells infused via the
portal vein were themselves OX-2.sup.+ (see description of
materials and methods above). However, identical data in FACS
analysis (FIG. 4) and Western Blots (FIG. 5), and from suppression
subtraction hybridization (FIG. 3), have been obtained in studies
in which we used irradiated whole spleen cells (OX-2.sup.- by FACS)
for pv infusion. This is consistent with the lack of evidence for
increased mRNA expression of OX-2 early (1-2 days) post transplant,
as noted above. Thus it seems most likely that an operationally
important "OX-2 signal" detected in the spleen of the pv immunized
mice can derive from new expression, rather than necessarily from
infused OX-2.sup.+ cells. In the absence of a polymorphic marker
for OX-2, however, it cannot be determined whether increased
expression is from donor or host cells (or both). Indeed, it is
perhaps somewhat surprising that the murine antibody to rat OX-2
cross-reacts in the fashion shown with murine OX-2. Definitive
analysis of the in vivo role of OX-2 awaits similar studies to
those above, using antibodies developed against the murine OX-2
homologue-these experiments are currently in progress. It is also
important to point out that while pv immunization led to only a
4-fold alteration in the absolute number of detectable OX-2.sup.+
NLDC.sup.+ cells in the spleen/liver (see text and FIG. 4),
nevertheless in the face of this 4-fold difference a clear
difference in OX-2 signals in Northern gels using RNA from pv vs iv
immunized mice (FIG. 3), along with evidence for a role for this
quantitative difference in the outcome of graft survival (FIG. 6)
were detected. Presumably these results reflect respectively the
limitation to the sensitivity of the Northern assay used, and some
function of the quantitation of "co-stimulation" occurring after
OX-2:OX-2 ligand interaction.
While there was some 50% homology of the predicted protein sequence
of murine OX-2 with murine B7-1, B7-2, CD28 and CTLA4 (Borriello et
al., 1997), antibodies to the latter molecules did not reverse the
prolonged graft survival and altered cytokine production following
pv immunization (FIG. 6, Table 2--see also (Castle et al., 1993)).
In fact these latter antibodies themselves, infused in the absence
of pv immunization, produced some of the same changes in cytokine
production induced by pv immunization (Table 2).
Example 2
Murine OX-2
This example describes the cloning and sequencing of murine MRC
OX-2.
A cDNA library was constructed from MLN cells derived from adult
C3H mice, preimmunized 5 days earlier with 10.times.106 allogeneic
B10.BR bone marrow-derived dendritic cells allogeneic cells by the
portal venous (pv) route, using a Cap Finder PCR cDNA library
construction kit (Clontech). The inventor had previously isolated,
using a PCR-Select CDNA subtraction hybridization kit (Clontech)
and RNAs obtained from pooled MLN of mice immunized by the pv route
or via the lateral tail vein (iv), a 350 bp amplicon which showed
over 98% homology with the 3' untranslated region of rat MRC OX-2
cDNA. Northern blot analysis confirmed that this amplicon detected
a differentially expressed product in RNAs prepared from iv vs pv
immunized mice. This amplicon was used to screen 5.times.105 clones
of the amplified library. The sequences of cDNA clones were
established with an Applied Biosystems 377 Automated Sequencer,
utilizing the Dye Terminator Cycle Sequencing method (Applied
Biosystems, Foster City, Calif.). The nucleotide sequence reported
in this paper has been submitted to the GenBank/EMBL Data Bank with
accession number AF004023.
The cDNA shown in FIG. 7 has an open reading frame of 837 base
pairs, and a deduced amino acid sequence (FIG. 8) of 248 amino
acids, of which 30 represent a cleaved leader sequence. The
predicted molecular weight of this, and the equivalent molecules in
rat and human, is approximately 25 kDa. The measured molecular
weight in rat thymocytes, where the molecule is highly
gylcosylated, is 47 kDa.
The murine MRC OX-2 shows some 92% and 77% homology overall at the
amino acid level with equivalent molecules in rat or human
respectively. As noted for the rat molecule, the sequence from a
203-229 seems likely to represent a membrane spanning domain
(highly hydrophobic region), while the region from 229-248 is
likely the intracytoplasmic region, with a stretch of highly basic
residues immediately C-terminal to position 229. Homology in the
combined transmembrane and C-terminal regions with rat and human
shows some 98% and 85% similarity respectively. As predicted from
membership in the Ig supergene family, there are a number of
conserved Cys residues forming the disulphide bonds between
b-strands of Ig-like domains, (21 and 91; 130 and 184
respectively); residue 91 was previously found to be the most
highly conserved among members of the immunoglobulin superfamily.
Homology between the N-terminal Ig-domain with rat and human,
versus the next Ig-domain, is 88% and 82%, or 97% and 73%
respectively. This relative concentration in variability between
rat and mouse in the V-terminal Ig-domain may be more
understandable when the ligand specificity for the molecules in
these species is clarified. Note that the presumed extracellular
portion of the molecule (1-202) contains a number of sites for
N-glycosylation which are preserved across species (44, 65, 73, 80,
94, 127, 130 and 151). This was previously reported for the rat
cDNA sequence, and inferred from the measured size of the expressed
material in rat thymocytes.
The intracytoplasmic region of the molecules has no sequence
identity with known signaling kinases, nor does it have the
well-described consensus sequence for the immunoreceptor tyrosine
activation motif (ITAM: DXXYXXLXXXXXXXYDXL). In addition, it lacks
typical SH2 or SH3 domains to serve as "docking sites" for adapter
molecules which might in turn co-opt other protein kinases in an
activation cascade. Accordingly the ligand-binding activity of the
extracellular domains presumably represent the biologically
important region of the molecule. Some possible functions
attributable to ligand interaction with OX-2 can be inferred from
other data in the literature. A homologous molecule, Ng-CAM, has
been reported to bind a protein-tyrosine phosphatase via N-linked
oligosaccharide residues, and protein tyrosine phosphatases are
known to play a key regulatory role in immune responses. More
recently ALCAM, another adhesion molecule member of the Ig
superfamily, the gene for which is located close to that for OX-2
on chromosome 3 in humans, has been shown to bind CD6 (a member of
the scavenger receptor cystein rich family, SRCR), and antibodies
to CD6 may themselves play a role in regulating immune
function.
Example 3
OX-2 Positive Cells Inhibit Type-1 Cytokine Production
The inventor has shown that hepatic mononuclear, non-parenchymal,
cells (NPC) can inhibit the immune response seen when allogeneic
C57BL/6 dendritic cells (DC) are incubated with C3H spleen
responder cells. Cells derived from these cultures transfer
increased survival of C57BL/6 renal allografts in C3H mice. The
inventor also found that increased expression of OX-2 on dendritic
cells was associated with inhibition of cytokine production and
renal allograft rejection. The inventor further explored whether
inhibition by hepatic NPC was a function of OX-2 expression by
these cells.
Fresh C57BL/6 spleen derived DC were cultured with C3H spleen
responder cells and other putative co-regulatory cells. The latter
were derived from fresh C3H or C57BL/6 liver NPC, or from C3H or
C57BL/6 mice treated for 10 days by intravenous infusion of human
Flt3 ligand (Flt3L). Different populations of murine bone-marrow
derived dendritic cells from cultures of bone marrow with
(IL-4+GM-CSF) were also used as a source of putative regulator
cells. Supernatants of all stimulated cultures were examined for
functional expression of different cytokines (IL-2, IL-4,
IFN.gamma., TGF.beta.). It was found that fresh C57BL/6 splenic DC
induced IL-2 not IL-4 production. Cells from the sources indicated
inhibited IL-2 and IFN.gamma. production, and promoted IL-4 and
TGF.beta. production. Inhibition was associated with increased
expression of OX-2 on these cells, as defined by semi-quantitative
PCR and FACS analysis. By size fractionation, cells expressing OX-2
were a subpopulation of NLDC145+ cells. This data implies a role
for cells expressing OX-2 in the regulation of induction of
cytokine production by conventional allostimulatory DC.
Materials and Methods
Mice: Male and female C3H/HeJ and B10.BR (H-2.sup.k/k), B10.D2
(H-2.sup.d/d) and C57BL/6 (H-2.sup.b/b) mice were purchased from
the Jackson laboratories, Bar Harbour, Maine. Mice were housed
5/cage and allowed food and water ad libitum. All mice were used at
8-12 weeks of age.
Monoclonal Antibodies: The following monoclonal antibodies (Mabs),
all obtained from Pharmingen (San Diego, Calif., USA) unless stated
otherwise, were used: anti-IL-2 (JES6-1A12; biotinylated,
JES6-5H4); anti-IL-4 (11B11, ATCC; biotinylated, BVD6-24G2);
anti-IFN.gamma. (R4-6A2, ATCC; biotinylated XMG1.2); anti-IL-10
(JES5-2A5; biotinylated SXC-1); PE anti-B7-1B7-2 (Cedarlane Labs,
Hornby, Ontario, Canada).
Rat anti-mouse OX-2 monoclonal antibodies were prepared by
Immuno-Precise Antibodies Ltd. (Victoria, BC, Canada) following
immunization of rats with a crude membrane extract of LPS
stimulated murine DC, followed by fusion with a non-secreting rat
myeloma parent cell line (YB2/3HI.P2.G11.16Ag.20). Hybridoma
supernatants were screened in ELISA using plates pre-coated with a
40-45 Kd preparation of DC extracts run on Western gels (Barclay,
A. N. 1981. Immunology 44:727; Barclay, A. N., and H. A. Ward.
1982. Eur. J. Biochem. 129:447). Positive clones were re-screened
using FACS analysis of CHO cells transduced with a cDNA clone
encoding full-length murine OX-2 (Chen, Z., H. Zeng, and R. M.
Gorczynski. 1997. BBA. Mol. Basis Dis. 1362:6-10). FITC-conjugated
F(ab')2 rabbit anti-rat IgG (non cross-reactive with mouse IgG)
from Serotec, Canada was used as second antibody. The Mab selected
for further analysis (M3B5) was grown in bulk in a CELLMAX system
(Cellco Inc., Germantown, Md.). A crude preparation of rat
immunoglobulin (30% saturated ammonium sulphate preparation) was
used as a control Ig.
In tissue culture assays where anti-cytokine Mabs were used to
confirm the specificity of the assay used 10 mg/ml of the relevant
Mabs was found to neutralize up to 5.0 ng/ml of the cytokine
tested.
NLDC145 (anti-mouse DC) was also obtained from Serotec. Recombinant
mouse IL-4 was a kind gift from Dr. L. Yang (The Toronto Hospital);
mouse rGM-CSF was purchased from Pharmingen. Recombinant human
Flt3L (derived from CHO cells) was a kind gift from Dr. A. B.
Troutt, Immunex Corp., Seattle, Wash., USA.
Renal Transplantation
Renal transplantation was performed essentially as described
elsewhere (Gorczynski, R. M. et al. 1994a. Transplantation
58:816-820). Animals were anesthetized with a combination of
halothane and nitrous oxide inhalation, using novogesic for post-op
analgesia. Orthotopic renal transplantation was performed using
routine procedures. In brief, Donor animals received 200 Units of
heparin, and kidneys were flushed with 2 ml of ice cold heparinized
physiological saline solution, prior to removal and transplantation
into recipient animals with left nephrectomy. The graft renal
artery was anastomosed to the recipient's abdominal aorta, and the
renal artery was anastomosed to the recipient's inferior vena cava.
The ureter was sewn into the recipient bladder using a small donor
bladder patch. All recipients received im injection with cefotetan
(30 mg/Kg) on the day of transplantation and for 2 succeeding days.
The remaining host kidney was removed 2 days after transplantation,
unless otherwise indicated. Treatment of recipients with pv
immunization, by monoclonal antibodies, or by oral immunization was
as described in individual studies.
Portal Vein and Oral Immunization
Portal vein and oral immunization was performed as described
earlier (Gorczynski, R. M. 1995a. Cell. Immunol. 160:224-231;
Gorczynski, R. M. et al. Transplantation 62:1592-1600). All animals
were anaesthetized with nembutal. A midline abdominal incision was
made and the viscera exposed. Cells were injected in 0.lml through
a superior mesenteric vein using a 30 gauge needle. After injection
the needle was rapidly withdrawn and hemostasis secured without
hematoma formation by gentle pressure using a 2.times.3 mm
gel-foam.
Bone-marrow derived dendritic cells (DC) for pv immunization were
obtained by culture of T depleted bone marrow cells in vitro with
rIL-4 and rGM-CSF (Gorczynski, R. M. et al. Transplantation
62:1592-1600). Staining with NLDC145 and FITC anti-rat IgG, or with
FITC anti-CD3 confirmed >95% NLDC145+ and <5% CD3+ cells at
day 10 of culture (Gorczynski, R. M. et al. Transplantation
62:1592-1600). These cells were washed and injected into mice or
used for mixed leucocyte cultures.
Preparation of Cells:
Spleen and bone marrow (Gorczynski, R. M. et al. Transplantation
62:1592-1600) cell suspensions were prepared aseptically from
individual mice in each experiment. Hepatic mononuclear
nonparenchymal cells (NPC) were isolated essentially as described
elsewhere (Gorczynski, R. M. 1994b. Immunology 81:27-35). Tissue
was first digested at 37.degree. C. for 45 min with a mixture of
collagenase/dispase, prior to separation (15 min at 17,000 rpm at
room temperature) over mouse lymphopaque (Cedarlane Labs).
Mononuclear cells were resuspended in a-Minimal Essential Medium
supplemented with 2-mercaptoethanol and 10% fetal calf serum
(aF10). Where cells were obtained from Flt3L injected mice, animals
were treated by iv injection of 10 mg/mouse Flt3L daily for 10
days. After enzyme digestion recovery of liver/spleen cells from
these mice was markedly increased compared with saline-injected
controls (120.times.10.sup.6, 390.times.10.sup.6 vs
7.times.10.sup.6 and 120.times.10.sup.6 respectively).
Cytotoxicity and Cytokine Assays:
In cultures used to assess induction of cytotoxicity or cytokine
production responder cells were stimulated with irradiated (2000R)
stimulator cells in triplicate in aF10. Supernatants were pooled
from replicate wells at 40 hrs for cytokine assays (below). No
reproducible differences in cytokine levels have been detected from
cultures assayed between 36 and 54 hrs of stimulation. In some
experiments the cultures received 1 mCi/well (at 72 hrs) of .sup.3
HTdR and proliferation was assessed by harvesting cells 14 hrs
later and counting in a well-type b-counter.
Where cytoxicity was measured cells were harvested and pooled from
equivalent cultures at 5 days, counted, and recultured at different
effector:target with .sup.51 Cr EL4 (H.sub.2.sup.b/b) or P815
(H2.sup.d/d) tumor target cells. Supernatants were sampled at 4 hrs
for assessment of specific cytotoxicity.
IL-2 and IL-4 activity were assayed by bioassay using the IL-2/IL-4
dependent cell lines, CTLL-2 and CT4.S respectively. Recombinant
cytokines for standardization of assays was purchased from Genzyme
(Cambridge, Mass.). IL-2 assays were set up in the presence of 11
B11 to block potential stimulation of CTLL-2 with IL-4; IL-4 assays
were set up in the presence of S4B6 to block IL-2 mediated
stimulation. Both the IL-2 and IL-4 assays reproducibly detected 50
pg of recombinant lymphokine added to cultures.
In addition, IL-2, IL-4, IFN.gamma. and IL-10 were assayed using
ELISA assays. For IFN.gamma. the assay used flat-bottomed Nunc
plates (Gibco, BRL) coated with 100 ng/ml R4-6A2. Varying dilutions
of supernatant were bound in triplicate at 4.degree. C., washed
.times.3, and biotinylated anti-IFN.gamma. (XMG1.2) added. After
washing, plates were incubated with streptavidin-horse radish
peroxidase (Cedarlane Labs, Hornby, Ontario), developed with
appropriate substrate, and OD.sub.405 determined using an ELISA
plate reader. Recombinant IFN.gamma. for standardization was from
Pharmingen. IL-10 was similarly assayed by ELISA, using JES5-2A5 as
a capture antibody and biotinylated SXC-1 as developing antibody.
rIL-10 for standardization was from Pepro Tech Inc. (Rocky Hill,
N.J.). Each assay detected 0.1 ng/ml cytokine. ELISA assays for
IL-2 and IL-4 used JES6-1A12 and 11B11 as capture antibodies, with
JAS6-5H4 or BVD6-24G2 as developing antibodies. Sensitivity of
detection was 20 pg/ml for each cytokine. Where checked the
correlation between bioassay and ELISA for IL-2 or IL-4 was
excellent (r>0.90). In all studies reported below, data are
shown from ELISA assays only. Where cytokine data are pooled from
several studies (e.g. FIGS. 14, 16, 17), absolute values of
cytokine production were obtained as above using commercial
recombinant cytokines to standardize the assays. In our hands,
supernatants from C3Hanti-C57BL/6 cultures, under the conditions
described, reproducibly contain 950.+-.200 and 80.+-.25 pg/ml IL-2
and IL-4 respectively.
Preparation of RNA:
Different sources of tissue from renal-grafted female mice
receiving DC and kidney allografts from male mice were harvested
for RNA extraction as described elsewhere (Gorczynski, R. M. 1995a.
Cell. Immunol. 160:224-231). The OD280/260 of each sample was
measured and reverse transcription performed using oligo (dT)
primers (27-7858: Pharmacia, USA). The cDNA was diluted to a total
volume of 100 ml with water and frozen at -70.degree. C. until use
in PCR reactions with primers for murine GAPDH, B7-1, B7-2 or OX-2.
The sense (S) and antisense (AS) primers were synthesized by the
Biotechnology Service Centre, Hospital for Sick Children, Toronto,
using published sequences. 5' primers were .sup.32 P end-labeled
for PCR and had comparable levels of specific activity after
purification by ethanol precipitation. 5ml cDNA was amplified for
35 cycles by PCR, and samples were analyzed in 12.5% polyacrylamide
gels followed by overnight (18 hrs) exposure for autoradiography.
In control studies, using H-Y primer sets, this technique reliably
detects H-Y mRNA from extracts of female spleen cells to which male
cells are added at a concentration of 1:105 (Gorczynski, R. M.
1995a. Cell. Immunol. 160:224-231; Gorczynski, R. M. et al.
Transplantation 62:1592-1600). Quantitative comparison of
expression of different PCR products used densitometric scanning of
the autoradiograms.
(SEQ.ID.NO.:10) GAPDH Sense: 5'TGATGACATCAAGAAGGTGGTGAAG3'
(SEQ.ID.NO.:11) GAPDH Antisense: 5'TCCTTGGAGGCCATGTAGGCCAT3'
(SEQ.ID.NO.:12) B7-1 Sense: 5'CCTTGCCGTTACAACTCTCC3'
(SEQ.ID.NO.:13) B7-1Antisense: 5'CGGAAGCAAAGCAGGTAATC3'
(SEQ.ID.NO.:14) B7-2 Sense: 5'TCTCAGATGCTGTTTCCGTG3'
(SEQ.ID.NO.:15) B7-2 Antisense: 5'GGTTCACTGAAGTTGGCGAT3'
(SEQ.ID.NO.:16) OX-2 Sense: 5'GTGGAAGTGGTGACCCAGGA3'
(SEQ.ID.NO.:17) OX-2 Antisense: 5'ATAGAGAGTAAGGCAAGCTG3'
Statistical Analysis:
In studies with multiple groups, ANOVA was performed to compare
significance. In some cases (as defined in individual
circumstances) pairwise comparison between groups was also
subsequently performed.
Results
Antigen Stimulation, in the Presence of Hepatic NPC, Induces
Development of a Cell Population Capable of Inhibiting
Proliferation and IL-2 Production on Adoptive Transfer:
In a previous manuscript (Gorczynski, R. M. et al.,
Transplantation. 66: 000-008) it was reported that C3H spleen cells
stimulated in the presence of syngeneic NPC and allogeneic
(C57BL/6) DC produced a cell population able to inhibit generation
of IL-2 from fresh spleen cells stimulated with C57BL/6 DC, and
capable of inhibiting C57BL/6 renal allograft rejection in vivo. In
order to ask whether this function of NPC was MHC restricted or
not, the following study was performed.
C57BL/6 (H2.sup.b/b) spleen cells were stimulated in vitro with
B10.BR (H2.sup.k/k) bone-marrow derived DC, in the presence/absence
of the following NPC: C57BL/6; B10.BR; B10.D2 (H2.sup.d/d). In
addition, control cultures were incubated with the NPC only.
Proliferation and IL-2/IL-4 production was measured in one aliquot
of these primary cultures. In addition, at 5 days, cells were
harvested from another set of the primary cultures, washed, and
2.times.10.sup.5 cells added to cultures containing
5.times.10.sup.6 fresh C57BL/6 spleen cells and B10.BR DC.
Proliferation and cytokine production was measured in these latter
cultures in standard fashion. Data pooled from three equivalent
studies are shown in panels A) and B) of FIG. 9.
FIG. 9 is a bar graph showing regulation of proliferation and
cytokine production following stimulation by allogeneic DC using
hepatic NPC in accordance with the methods described herein. In
panel A) cultures were initiated with 5.times.10.sup.6 C57BL/6
responder spleen cells alone (group 1), or with 2.times.10.sup.5
B10.BR DC (group 2). Further groups (3-5, and 6-8 respectively)
contained C57BL/6 responder cells and 2.times.10.sup.5 NPC from
either C57BL/6, B10.D2 or B10.BR respectively (3-5) or these same
NPC and B10.BR DC (6-8). Data show mean proliferation and cytokine
production from triplicate cultures in three separate studies. In
panel B) data show proliferation and cytokine production from
cultures of 5.times.10.sup.6 C57BL/6 responder spleen cells
stimulated in triplicate with 2.times.10.sup.5 B10.BR DC alone, or
with the addition also of 2.times.10.sup.5 cells harvested from the
cultures shown in the upper panel. Again data represent arithmetic
means of 3 separate experiments. *p<0.05 compared with control
cultures (far left in each panel).
There are a number of points of interest. As previously documented,
addition of NPC syngeneic with spleen responder cells (C57BL/6 in
this case) to cells stimulated with allogeneic (B10.BR) DC led to
decreased proliferation and IL-2 production from those responder
cells compared with cells stimulated by DC alone (compare groups 6
and 2 of upper panel of FIG. 9, panel A). IL-4 production in
contrast was enhanced. NPC alone, whether syngeneic or allogeneic
to the responder cells, produced no obvious effect (groups 3-5,
panel A) of FIG. 9). Furthermore, cells from primary cultures
receiving the DC+NPC mixture were able to inhibit proliferation and
IL-2 production (while promoting IL-4 production) from fresh spleen
cells stimulated in secondary cultures with the same (B10.BR) DC
(see panel B) of FIG. 9). However, data in this Figure make another
important point. The same inhibition of proliferation/IL-2
production in primary cultures was seen using either B10.BR NPC
(MHC matched with the DC stimulus-group 8, panel A) of FIG. 9) or
with third-party B10.D2 NPC (MHC-mismatched with both spleen
responder cells and allogeneic stimulator DC-group 7, panel A) of
FIG. 9). Again no obvious effect was seen in cultures stimulated
with B10.BR or B10.D2 NPC alone (groups 4 and 5). Finally, cells
taken from primary cultures stimulated with DC and NPC from either
B10.BR or B10.D2 could also inhibit proliferation/IL-2 production
from secondary C57BL/6 spleen cell cultures stimulated with B10.BR
DC-again cells taken from primary cultures with NPC alone produced
no such inhibition (see panel B) of FIG. 9). Thus the inhibition of
proliferation/IL-2 production and enhancement of IL-4 production
seen in primary cultures, as well as the induction of suppression
measured in secondary cultures, all induced by NPC, are not
MHC-restricted.
Specificity of Inhibition/Suppression Induced by Hepatic NPC:
One interpretation of the data shown in FIG. 9 and elsewhere is
that NPC deliver a signal to DC-stimulated cells which is distinct
from the antigen-signal provided by the DC themselves (and is MHC
non-restricted). This signal modulates the antigen-specific signal
provided by the DC. In order to assess the antigen-specificity of
the immunoregulation described in FIG. 9, the following experiment
was performed.
C57BL/6 spleen responder cells were stimulated with B10.D2 or
B10.BR bone marrow-derived DC, in the presence/absence of NPC from
B10.BR or B10.D2 mice. Proliferation and cytokine production was
measured in aliquots of these cultures as before. In addition,
further aliquots of cells harvested from these primary cultures
were added to cultures of fresh C57BL/6 spleen cells stimulated
with B10.BR (panel B)--FIG. 10) or B10.D2 (panel C)-FIG. 10) DC.
Again proliferation and cytokine production was measured. Data
pooled from three such studies are shown in FIG. 10.
FIG. 10 shows specificity of inhibition of proliferation of
cytokine production by hepatic NPC (see FIG. 9 and description of
FIG. 9 for more details). In panel A), 5.times.106 C57BL/6 spleen
cells were stimulated in triplicate for 3 days with
2.times.10.sup.5 B10.BR or B10.D2 DC, with/without 2.times.10.sup.5
NPC derived from B10.D2 or B10.BR mice. Data shown are arithmetic
means of 3 repeat studies. In panels B) and C), fresh C57BL/6
responder spleen cells were cultured in triplicate with either
B10.BR DC (panel B), or B10.D2 DC (Panel C), with/without
2.times.10.sup.5 additional cells from the primary cultures (groups
1-6 in panel A). Again data represent arithmetic means of
proliferation/cytokine production from 3 studies. *p<0.05
compared with control cultures (far left in each panel).
Data from the primary cultures (panel A)) recapitulates the
observations made in FIG. 9, and show that NPC inhibit
proliferation and IL-2 production from DC-stimulated responder
cells in an antigen and MHC-unrestricted fashion. However, the data
in panels B) and C) of this figure show clearly that adoptive
transfer of inhibition using cells from these primary cultures
occurs in an antigen-restricted fashion, dictated by the
antigen-specificity of the DC used in the primary cultures, not of
the NPC used for induction of suppression. These auxiliary cells in
the NPC population thus have a functional property of being
"facilitator cells for induction of suppression". Note that in
other studies (data not shown) where the final assay system
involved measuring cytotoxicity to allogeneic target cells, a
similar inhibition of lysis (rather than cytokine production) was
seen using cells harvested from primary cultures stimulated with DC
and hepatic NPC (see Gorczynski, R. M., et al. 1998a.
Transplantation. 66: 000-008).
Hepatic Cell Preparations from Flt3L Treated Mice are a Potent
Source of DC and "Facilitator" Cells:
It has been reported at length that pv infusion of alloantigen, or
iv infusion of liver-derived allogeneic mononuclear cells induces
operational unresponsiveness in recipient animals (Gorczynski, R.
M. 1995a. Cell. Immunol. 160:224-231; Gorczynski, R. M. et al.
Transplantation 62:1592-1600; Gorczynski, R. M. et al. 1994a.
Transplantation 58:816-820.; Gorczynski, R. M., and D. Wojcik.
1992. Immunol. Lett. 34:177-182; Gorczynski, R. M. et al. 1995b.
Transplantation. 60:1337-1341). The total hepatic mononuclear cell
yield from normal mice is of the order of 5.times.10.sup.6
cells/mouse. In order to increase the yield, and explore the
possibility that the liver itself might be a source both of
allostimulatory DC and "facilitator" cells 2 C57BL/6 mice were
exposed for 10 days to daily iv infusions of 10 mg/mouse human
CHO-derived Flt3L, a known growth factor for DC (Steptoe, R. J. et
al. 1997. J Immunol. 159:5483-5491). Liver tissue was harvested and
pooled from these donors and mononuclear cells prepared as
described in the Materials and Methods section above (mean
130.times.10.sup.6 cells/donor). These cells were further subjected
to sub-fractionation by size using unit gravity sedimentation
techniques (Miller, R. G., and R. A. Phillips. 1969. J. Cell. Comp.
Physiol. 73:191-198). A typical size profile for recovered cells is
shown in FIG. 11 (one of 3 studies).
FIG. 11 shows OX-2 expression in a subpopulation of NPC. It is a
sedimentation analysis (cell profile) and FACS analysis of cells
isolated at 10 days from Flt3L-treated C57BL/6 mice. Two C57BL/67
mice received 10 mg/mouse Flt3L iv daily for 10 days. Hepatic NPC
were sedimented for 3 hrs at 4.degree. C., and the fractions shown
collected (Fxs 1-4 with sedimentation velocities 2.5-3.8, 3.8-5.1,
5.1-6.4 and 6.4-8.0 mm/hr respectively). Aliquots of the cells were
stained in triplicate with the Mabs shown. The remainder of the
cells were used as in FIGS. 12-14. Data are pooled from 3
studies.
In these same studies cells isolated from the various fractions
shown in FIG. 11 were tested as follows. Firstly, cells were
stained with FITC-labeled Mabs to B7-1, B7-2, NLDC145 and rat
anti-mouse OX-2 (M3B5) with FITC anti-rat IgG as second antibody.
In addition, mRNA extracted from the different cell samples were
assayed by PCR for expression of GAPDH, B7-1, B7-2 and OX-2. Data
are shown in FIG. 11 (pooled from 3 separate studies) and FIG. 12
(representative PCR data from one experiment).
FIG. 12 shows PCR detection of B7-1, B7-2 and OX-2 in hepatic NPMC.
It is a PCR analysis for mRNA expression of OX-2, B7-1 and B7-2 in
various hepatic NPC cell fractions isolated from Flt3L treated mice
(see FIG. 11). Data are representative from 1 of 3 studies.
Further aliquots of the cells were used to stimulate fresh C3H
spleen responder cells in culture. Proliferation and cytokine
assays were performed as before (see FIG. 9), and in addition cells
were taken from these primary cultures and added to fresh secondary
cultures of C3H spleen responder cells and C57BL/6 bone
marrow-derived DC. Again proliferation and cytokine production was
assayed from these secondary cultures. Data pooled from 3 studies
of this type are shown in FIG. 13 (panels A) and B).
FIG. 13 shows that hepatic NPMC from Flt3L treated mice results
IL-2 and IL-4 production. Stimulation of proliferation/cytokine
production by NPC from Flt3L treated mice, and inhibition of the
same (where stimulation is induced by a separate population of DC)
is a function of different cell populations. (See text and FIGS.
11-12 for more details.) Hepatic NPC fractions were derived from
Flt3L treated C57BL/6 mice and were used to stimulate C3H spleen
cells in triplicate cultures, alone or in the presence of
bone-marrow derived C57BL/6 DC (see panel A). Data show arithmetic
means for proliferation/cytokine production from 3 experiments. In
addition, cells harvested from these primary cultures were added to
fresh C3H spleen cells stimulated with C57BL/6 DC (panel B), and
again proliferation/cytokine production assayed. *p<0.05
compared with control groups (far left of panel).
Finally, cells from the various fractions were infused iv into
2/group C3H mice which also received C57BL/6 renal allografts as
antigen challenge. Spleen cells were harvested from these
individual mice 10 days after transplantation and restimulated in
culture with C57BL/6 or B10.D2 DC, again with cytokines measured at
40 hrs (see FIG. 14).
FIG. 14 is a bar graph of cytokines produced from cells from C3H
mice with C57BL/b renal allografts and NPC from Flt3 treated
C57BL/6 donors. OX-2.sup.+ NPC infused iv into renal transplant
allograft recipients leads to polarization of cytokine production
(to IL-4, IL-10 and TGF.beta.) in spleen cells harvested from those
mice and restimulated in vitro. Fractions of NPC from Flt3L treated
C57BL/6 mice (from FIG. 11) were infused iv into 2/group C3H
recipients, receiving C57BL/6 renal allografts (along with CsA) in
standard fashion (see Materials and Methods). Mice were sacrificed
14 days after transplantation and spleen cells stimulated in vitro
in triplicate with C57BU6 DC stimulator cells. Cytokines were
assayed in the supernatants of these cultures at 60 hrs. Data show
arithmetic means pooled from cultures in 3 studies of this type.
*p<0.05 compared with control groups (far left-no NPC
infused).
Data in FIG. 11 show that distinct subpopulations of
slow-sedimenting cells express OX-2 in the cells harvested from
Flt3L treated mice, when compared with cells expressing B7-1 and/or
B7-2. In general expression of OX-2 and B7-2 occured in equivalent
subpopulations. Faster-sedimenting cells (Fx 3 and 4 in FIG. 11),
while staining for NLDC145, were positive by fluorescence mainly
for B7-1, not B7-2 or OX-2. Similar conclusions were reached both
by FACS analysis of cell populations (FIG. 11), and by PCR analysis
of mRNA (FIG. 12).
When the functional capacity of these different cell populations
was investigated (FIGS. 13 and 14) it was found that optimal direct
stimulation (or proliferation and IL-2 production) was seen from
B7-1 expressing cells (Fxs 3 and 4 in panel A) of FIG. 13), while
only OX-2 expressing cells (Fxs 1 and 2 in FIGS. 11 and 12) were
capable of producing the inhibitory effects defined earlier (FIGS.
9 & 10) in the two-stage culture system (panel B) in FIG. 13).
These same cells (Fxs 1 and 2) were in turn able, after iv
infusion, to polarize cells from mice given renal allografts to
produce predominantly IL-4, IL-10 and TGF.beta. production on
restimulation in vitro (FIG. 14). These data are consistent with
the notion that after FltL treatment of mice expansion of a
population of immunostimulatory DC occurs within the liver, which
also contains another distinct population of (facilitator) cells
which promote immunoregulation.
Evidence that Cell Populations with "Facilitator" Activity from the
Liver of Flt3L Treated Mice Prolong Graft Survival in vivo:
Since it has been reported elsewhere that there is a good
correlation between treatments (such as pv immunization) which
decrease IL-2 production and increase IL-4 production from
restimulated cells and prolongation of graft survival (Gorczynski,
R. M., and D. Wojcik. 1994. J. Immunol. 152:2011-2019; Gorczynski,
R. M. 1995a. Cell. Immunol. 160:224-231; Gorczynski, R. M. et al.
Transplantation 62:1592-1600), and that increased expression of
OX-2 is also independently associated with increased graft survival
after pv immunization (Gorczynski, R. M. et al. 1998b.
Transplantation. 65:1106-1114), the next question was whether cells
isolated from Flt3L treated mice which induced inhibitory function
in vitro (see FIGS. 9, 10 and 13), and expressed increased amounts
of OX-2 (FIGS. 11, 12) were themselves capable of promoting
increased graft survival in vivo.
Groups of 2 C57BL/6 mice received iv infusions of 10 mg/mouse Flt3L
for 10 days as before. Cells were isolated from the liver by enzyme
digestion, and fractionated by unit gravity sedimentation. 4 pools
of cells were recovered, and an aliquot stained as before in FACS
with anti-OX-2. Groups of 2 C3H mice received 10.times.10.sup.6
cells iv from the 4 separate pools. A control group received saline
injections only. Over the next 48 hrs all mice received C57BL/6
renal transplants. All mice received CsA (10 mg/Kg) on the day of
renal transplantation. Data in FIG. 15 are pooled from 3 studies of
this type (representing 6 mice/group), and show the animal survival
in these 5 different groups.
FIG. 15 shows NPC from Flt3L treated C57BL/6 mice, infused iv into
recipient C3H mice, inhibit C57BL/6 renal allograft rejection. Two
mice groups received the different subpopulations of NPC derived
from Flt3L treated mice shown in FIGS. 11 and 12. Fxs 1 and 2 were
OX-2+. Mice received C57BL/6 renal allografts within 48 hrs along
with CsA (see Materials and Methods). Animal survival was followed
as an end point. Data shown are pooled from 3 studies (6
mice/group). *p<0.05 compared with mice receiving CsA only (
).
It is quite clear from this Figure that only hepatic cells
expressing OX-2 (Fxs 1 and 2--see FIGS. 11 and 12) were capable of
promoting increased graft survival after iv infusion. Comparison of
these data with those in FIG. 13 confirm that these cell
populations were also those identified, using a 2-stage culture
assay system, as cells with functional "facilitator" activity (see
also FIGS. 9 and 10). There was no significant difference in
survival between groups receiving NPC-Fx1 or NPC-Fx2 in this
experiment, in keeping with relatively equivalent levels of OX-2
expression in these fractions (FIG. 11).
Anti-OX-2 Monoclonal Antibody in vitro Reverses Regulation Induced
by Hepatic NPC:
A final study was directed to whether anti-OX-2 monoclonal antibody
M3B5, added to cultures of C3H spleen responder cells, allogeneic
(C57BL/6) DC and NPC from C57BL/6 mice, could prevent the
inhibition of IL-2 production in primary cultures, and the
development of cells able to inhibit such cytokine responses from
freshly stimulated responder cells in secondary cultures (see FIGS.
9, 10 and 13). Data in FIGS. 16 and 17 are pooled from 3 studies of
this type.
FIG. 16 is a bar graph showing the effect of anti B7-1; B7-2; or
OX-2 on primary allostimulation. It shows that anti-OX-2 Mab
increases IL-2 cytokine production in vitro after stimulation of
C3H responder spleen cells with C57BL/6 DC. Subgroups of cultures
contained the Mabs shown. Cytokines were assayed at 60 hrs. All
data represent arithmetic means pooled from 3 repeat studies.
*p<0.05 compared with control group (far left).
FIG. 17 is a bar graph showing that anti-OX-2 reverses inhibition
by NPC. It shows that anti-OX-2 Mab inhibits development of
immunoregulatory cells in vitro following incubation with hepatic
NPC. C3H responder spleen cells were incubated in triplicate with
C57BL/6 DC along with NPC (see FIGS. 9 and 10). Subgoups of these
cultures contained the Mabs shown. Cytokines were assayed in
cultures at 60 hrs (panel A). In addition, cells were harvested
from all groups, washed and added to fresh C3H responder spleen
cells and C57BL/6 DC (panel B). Cytokines in these groups were
assayed 60 hrs later. All data represent arithmetic means pooled
from 3 repeat studies. *p<0.05 compared with control group from
cultures of NPC with no monoclonal antibodies (far left in
Figure)--see also FIG. 16.
Primary cultures were of two types, containing C3H responder spleen
cells and C57BU6 DC alone (FIG. 16), or the same mixture with added
C57BL/6 NPC (FIG. 17). Subsets of these cultures contained in
addition either 5 mg/ml of anti-B7-1, anti-B7-2 or anti-OX-2.
Supernatants from responder cells stimulated in the presence of DC
only were assayed after 60 hrs for cytokine production (FIG. 16).
For the primary cultures incubated with both DC and NPC,
supernatants were harvested at 60 hrs and tested for cytokine
production (FIG. 17A). In addition, cells were harvested after 5
days, washed, and added to secondary cultures of fresh C3H
responder cells with fresh C57BL/6 DC. No monoclonal antibodies
were added at this second culture stage. Data for cytokine
production these secondary cultures are shown in FIG. 17B.
Addition of anti-B7-1 or anti-B7-2 to DC stimulated spleen cultures
led to inhibition of cytokine production (FIG. 16), while in
contrast anti-OX-2 monoclonal antibody led an increase in IL-2
production in these primary cultures (FIG. 16). We have reported
similar findings elsewhere (Ragheb et al-submitted for
publication). Interestingly, anti-OX-2 abolished the inhibition of
cytokine production caused by NPC in these primary cultures (FIG.
17A--see also FIGS. 9, 10 and 13). In addition, anti-OX-2 prevented
the functional development of a cell population capable of
transferring inhibition of cytokine production to freshly
stimulated spleen cells (FIG. 17B).
Discussion
There is considerable theoretical as well as practical interest in
understanding the mechanism(s) by which a state of antigen specific
tolerance can be induced in lymphoid populations. Limits to the
effective induction of tolerance represent a major challenge to
more successful allo (and xeno) transplantation, to name but one
example (Akatsuka, Y., C. Cerveny, and J. A. Hansen. 1996. Hum.
Immunol. 48:125-134). Significant efforts have been invested into
exploring how pre- (or peri-) transplant donor-specific
immunization might produce such a state (Qian, J. H. et al. 1985.
J. Immunol. 134:3656-3663; Kenick, S., et al. 1987. Transpl. Proc.
19:478-480; Gorczynski, R. M. 1992. ImmunoL Lett. 33:67-77; Thelen,
M., and U. Wirthmueller. 1994. Curr. Opin. Immunol. 6:106-112;
Akolkar, P. N. et al. 1993. J. Immunol. 150 (April 1):2761-2773;
Ahvazi, B. C. et al. J. Leu. Biol. 58 (1):23-31; Albina, J. E. et
al. 1991. J. Immunol. 147:144-152). There is good evidence that
portal venous (pv) immunization somehow leads to tolerance
induction, and this immunoregulation can apparently be monitored by
following changes in cytokine production from host cells, with
decreased production of IL-2, IL-12 and IFN.gamma., and increased
IL-4, IL-10, IL-13 and TGF.beta. (Thelen, M., and U. Wirthmueller.
1994. Curr. Opin. Immunol. 6:106-112; Gorczynski, R. M. et al.
1998a. Transplantation. 66: 000-008). Which, if any, of these
cytokine changes is directly and causally implicated nevertheless
remains obscure.
Further analysis of the cell population able to induce tolerance
after pv immunization led to the somewhat paradoxical observation
that donor dendritic (DC) cells represented an excellent tolerizing
population (Gorczynski, R. M. 1995a. Cell. Immunol. 160:224-231;
Gorczynski, R. M. et al. Transplantation 62:1592-1600). Since
antigen-pulsed DC are conventionally thought of as representing an
optimal immunizing regime, the mechanism(s) activated following DC
pv immmunization which led to tolerance (Banchereau, J., and R. M.
Steinman. 1998. Nature. 392:245-252) was of interest. It is already
clear that DC themselves represent an extremely heterogeneous
population, in terms of origin, cell surface phenotype, turnover in
vivo and possibly function (Salomon, B. et al. 1998. J. Immunol.
160:708-717; Leenen, P. J. M. et al. 1998. J. Immunol.
160:2166-2173). In the mouse lymph node at least 3 discrete
populations were identified, one of which comprised small
CD8a.sup.+ NLDC145.sup.+ cells, likely of lymphoid origin, with an
immature phenotype, and whose numbers were profoundly increased
(100.times.) following Flt3L treatment in vivo (Salomon, B. et al.
1998. J. Immunol. 160:708-717) (administration of the latter has
been reported to lead to proliferation of dendritic cells and other
cells of hematopoietic origin (Maraskovsky, E. et al. 1996. J.
Exptl. Med. 184:1953-1962)). These cells resembled the
interdigitating DC found in the T cell areas of the splenic white
pulp, and have been implicated in regulation of immunity induced by
other (myeloid derived) DC (Salomon, B. et al. 1998. J. Immunol.
160:708-717; Kronin, V. et al. 1996. J. Immunol. 157:3819-3827;
Suss, G., and K. Shortman. 1996. J. Exptl. Med. 183:1789-1796).
A variety of other studies have indicated that the induction of
immunity (vs tolerance) following antigen presentation was
intrinsically dependent upon the co-existence of other signaling
ligands at the surface of DC (interacting with appropriate
counter-ligands on the surface of other cells (e.g. stimulated T
cells)) (Larsen, C. P. et al. 1994. J. Immunol. 152:5208-5219;
Lenschow, D. J. et al. 1996. Annu. Rev. Immunol. 14:233-258;
Larsen, C. P., and T. C. Pearson. 1997. Curr. Opin. Immunol.
9:641-647). It was speculated that infusion of DC via the portal
vein induced tolerance by co-opting another cell population,
distinguishable by expression of unique cell surface ligands, whose
biological function was to facilitate induction of tolerance, not
immunity, when antigen was presented in association with otherwise
immunogenic DC. Some preliminary evidence supporting this
hypothesis was recently reported (Gorczynski, R. M. et al. 1998a.
Transplantation. 66: 000-008). Herein, this is referred to as a
facilitator cell. Moreover, because pv immunization has been shown
to be associated with increased expression of a novel molecule,
OX-2, previously reported to be expressed on DC (Barclay, A. N.
1981. Immunology 44:727; Barclay, A. N., and H. A. Ward. 1982. Eur.
J. Biochem. 129:447; Chen, Z. et al. 1997. BBA. Mol. Basis Dis.
1362:6-10; Gorczynski, R. M. et al. 1998b. Transplantation.
65:1106-1114), it was predicted that this molecule would in fact
serve as a "marker" for the hypothetical facilitator cell
described. Experiments reported herein are consistent with such a
hypothesis.
It is here shown that within the hepatic NPC population there is a
subset of cells able to inhibit stimulation by allogeneic DC in a
non-MHC restricted fashion (see FIGS. 9 and 10), and able to induce
the development of an antigen-specific immunoregulatory cell
population in vitro (see FIGS. 9 and 10). The non-MHC-restricted
nature of this "facilitator" cell interaction indicates that it
functions by providing an accessory signal (a regulatory not a
co-stimulatory signal) to the DC which stimulate T cells in the
allogeneic mixed leukocyte reaction described, in a fashion
analogous to the original description of costimulatory interactions
(Jenkins, M. K. et al. 1988. J. Immunol. 140:3324-3329). As a
result the stimulated lymphocytes alter their cytokine production
profile (with decreased IL-2 production and proliferation), and
become able to regulate the immune response seen from freshly
stimulated lymphocytes (see panel B in FIGS. 9 and 10). Most
interestingly, following expansion of DC in vivo by Flt3L
treatment, it is shown that in fact the liver itself contains both
an immunostimulating population (large cells by velocity
sedimentation analysis), and this putative "facilitator" cell
population (see FIGS. 11-15). Furthermore, the latter biological
activity resides within a slow-sedimenting (small size)
NLDC145.sup.+ cell population expressing preferentially both cell
surface B7-2 and OX-2 (see FIGS. 11 and 12). When it was
investigated whether this same population of cells was active in
vivo in regulating graft tolerance, it was found again that after
prior Flt3L treatment, the liver contained a population of cells
which transferred increased renal graft acceptance (FIG. 15) and in
parallel altered the cytokine production profile of immunized mice
towards increased IL-4 and TGF.beta., and decreased IL-2 and
IFN.gamma. production (FIG. 14).
In a final attempt to explore the role for OX-2 expression itself
in this regulatory function, fresh spleen cells were stimulated
with DC alone or in the presence of anti-B7-1, anti-B7-2 or
anti-OX-2. Note that other studies (data not shown) have confirmed
that even the bone-marrow derived DC used contains small numbers of
OX-2.sup.+ cells (RMG-unpublished). Unlike anti-B7-1 and anti-B7-2
which decreased cytokine production, a result in keeping with the
hypothesized role for these as costimulator molecules (Hancock, W.
W. et al. 1996. Proc. Natl. Acad. Sci. USA. 93:13967-13972;
Freeman, G. J. et al. 1995. Immunity. 2:523-532; Kuchroo, V. K. et
al. 1995. Cell. 80:707-718), anti-OX-2 produced a small but
significant (1.7-2.5 fold in three studies) increase in IL-2
production in this system (FIG. 16). Most important, however,
inclusion of anti-OX-2 Mab in a system where exogenous
"facilitator" cells were added (from NPC), blocked completely the
induction of inhibition normally seen in such cultures (FIGS. 9 and
10; compare with lower panel of FIG. 17). These data are consistent
with the concept that OX-2 delivers a regulatory, not a
costimulatory, signal in this situation.
How does the present data fit within the evolving framework of
understanding in the heterogeneity of DC? As noted above, there has
been speculation that a separate population of CD8a.sup.+
NLDC145.sup.+ DC of lymphoid origin which proliferates in response
to Flt3L, might be responsible for immunoregulation. Other data
have implicated IL-10 as a cytokine which might modify
development/maturation of DC into a population expressing increased
amounts of B7-2 and capable of inducing tolerance (Steinbrink, K.
et al. 1997. J Immunol. 159:4772-4780). The role of regulation of
expression of Fas as a controlling feature in this regard is
unexplored (Suss, G., and K. Shortman. 1996. J. Exptl. Med.
183:1789-1796). The data disclosed herein is the first to implicate
another molecule, OX-2, in the delivery of a tolerizing signal,
perhaps in association with alterations in expression of B7-2, Fas
etc. It is intriguing that while there is clearly a key role for
intra-thymic DC in the regulation of self-tolerance (Banchereau,
J., and R. M. Steinman. 1998. Nature. 392:245-252), natural
expression of OX-2 was initially first described on thymic DC (as
well as within the brain) (Barclay, A. N. 1981. Immunology
44:727)--there is as yet no evidence to suggest that this
represents a functionally relevant expression for OX-2 in this
location. However, other independent data have also implied an
immunoregulatory role for OX-2 expression, again as assayed by
altered cytokine production in vitro from cells stimulated in the
presence/absence of expressed OX-2 (Borriello, F. et al. 1997. J.
Immuno. 158:4548).
It has been reported that following pv immunization there is a
measureable expansion in numbers of populations of
.gamma..delta.TCR.sup.+ cells capable of adoptive transfer of
increased graft survival to naive recipients (Gorczynski, R. M. et
al. 1996c. Immunology. 87 (3):381-389). Little is known concerning
the nature of the antigen recognized by these cells, and why, as a
population, their numbers are preferentially increased following pv
immunization. It is speculated that this may be explainable
ultimately in terms of a differential susceptibility of
.gamma..delta.TCR.sup.+ vs .alpha..beta.TCR.sup.+ cells to
immunoregulatory signals delivered following OX-2 expression.
In conclusion, the inventor has reported for the first time that
functional heterogeneity in the DC pool may be understandable in
terms of differential expression of OX-2 on the cell surface.
Expression of this molecule seems to give cells the capability to
induce immunoregulation, increased renal graft survival (and
altered cytokine production both in vivo and in vitro). The present
invention suggests that such OX-2 expressing cells are referred to
as "facilitator" cells (for tolerance induction). Indeed, using
FITC-OX-2:Fc, it was possible to show binding to putative CD200R on
>80% of activated gamma-delta T cells, whereas <20% of
alpha-beta T cells stained.
Example 4
Preparation of Murine Antibodies
Mouse and rat hybridomas to a 43 Kd molecule expressed in the
thymus, on a subpopulation of dendritic cells, and in the brain, in
mammalian tissue derived from mouse, rat and human were prepared.
Using CHO cells transiently transfected with adenovirus vector(s)
expressing a cDNA construct for the relevant OX-2 gene, the
monoclonal antibodies (Mabs) detect a molecule encoded by this
construct (rat OX-2 (rOX-2), mouse OX-2 (mOX-2) and human OX-2
(huOX-2) respectively). Furthermore, at least some of the anti-rat
Mabs detect determinants expressed on the murine OX-2 molecule.
Materials and Methods
Antigen preparation from tissues and Western blotting were
performed as described in Gorczynski et al., Transplantation, 1998,
65:1106-1114:
Spleen cells (human samples were obtained from cadavers at the time
of organ retrieval for transplantation) were used for preparation
of dendritic cells/macrophages. Tissue was digested with a mixture
of collagenase and dispase and centrifuged over lymphopaque. Cells
were adhered for 2 hr at 37.degree. C., washed vigorously, and
incubated for 14 hr at 37.degree. C. Dendritic cells were isolated
as non-adherent cells (Gorczynski et al., Transplantation, 1996.
62:1592-1600). Routine staining of mouse splenocytes with NLDC-145
and FITC anti-rat IgG, or FITC-MAC-1 before and after overnight
incubation produced the following staining pattern in these
adherent cells: 8%.+-.2%, 90%.+-.1 1% and 92%.+-.9%, 9%.+-.3%
respectively. The crude (non-adherent) dendritic cell preparation
was extracted with lysis buffer, titred to a protein concentration
of 10 mg/ml, and used for immunization. Some of the same material
was used subsequently in screening ELISAs (below).
When brain tissue was used in Western gel analysis, whole tissue
extract was electrophoresed in 12% SDS-PAGE and transferred to PVDF
membranes (Novex Co., San Diego, Calif.). Putative anti-OX-2 Mabs
were used as test reagent, with isotypic antibodies (negative in
ELISA tests) used as controls. Membranes were developed using
either anti-rat or anti-mouse horse radish peroxidase and
appropriate substrate.
Immunization and Production of Mabs:
Four female BALB/c mice were initially immunized by intraperitoneal
injections with lmg of human or rat dendritic antigen in Complete
Freundis Adjuvant. Three subsequent boosts were administered as
above, spaced at 3 week intervals, with Incomplete Freundis
Adjuvant. When the serum titre had risen more than 10-fold from a
pre-immune serum sample, as determined by ELISA, the 2 highest
responders were boosted intravenously. Three days later the donor
mice were sacrificed and the spleen cells were harvested and
pooled. Fusion of the splenocytes with X63-Ag8.6.5.3 BALB/c
parental myeloma cells was performed as previously described
(Kohler, G. and C. Milstein. 1975. Nature. 25: p. 256-259), except
that one-step selection and cloning of the hybridomas was performed
in 0.8% methylcellulose medium (Immuno-Precise Antibodies Ltd.,
Victoria, BC). This proprietary semi-solid medium allows HAT
selection and cloning in a single step and eliminates the
overgrowth of slower growing desirable clones by faster growing,
perhaps undesirable, hybridomas. Clones were picked and resuspended
in wells of 96-well tissue culture plates in 200 ml of D-MEM medium
containing 1% hypoxanthine/thymidine, 20% Fetal Bovine serum,1%
OPI, and 1.times.106/ml BALB/c thymocytes. After 4 days, the
supernatants were screened by ELISA for antibody activity on plates
coated with the immunizing antigen. Putative positive hybridomas
were re-cloned by limited dilution cloning to ensure monoclonality
and screened in FACS on extracts prepared from brain tissue
(below).
For the production of rat mAbs, 2 Fisher rats were immunized as
above with mouse antigen. Essentially the same procedure was
followed, except the parental cell line used for the fusion was
YB2/0.
ELISA and FACS Analysis of Putative Mabs:
ELISA assays used polystyrene plates pre-coated with 100 ng/ml
poly-L-lysine, followed by overnight incubation with the crude
dendritic cell antigen (used for immunization) at 10 mg/ml. Wells
were developed after binding of hybridoma superntatants using the
anti-rat/anti-mouse horse radish peroxidase antibodies above and
plates were analysed in an automatic ELISA plate reader (TiterTek
Multiskan, MCC/340, FlowLabs, Mississauga, Ontario, Canada).
FACS analysis was performed using putative anti-OX-2 Mabs and the
following cells. Fresh peripheral blood leucocytes (PBL), isolated
over rat/mouse lymphopaque (Cedarlane laboratories) or
Ficoll-Hypaque (human); fresh spleen dendritic cells (isolated
after adherence and overnight incubation, as above); and CHO cells
transduced with viral vectors engineered to contain a single copy
of a cDNA inserted into the notl/bamHl sites, encoding the relevant
species-specific OX-2, as per published sequences (Chen, Z. et al.
1997. BBA. Mol. Basis Dis. 1362:6-10; McCaughan, G. W., et al.
1987. Immunogenetics. 25: p. 133-135), or with control vector
alone. FITC anti-mouse (or anti-rat) IgG was used as secondary
antibody.
Mixed Leucocyte Reactivity (MLR) and Cytokine Production:
Allogeneic MLR cultures, using 1:1 mixtures of 2.5.times.10.sup.6
responder PBL and mitomycin C treated stimulator PBL, were set up
in 24-well culture plates in 1 ml of aMEM medium supplemented with
10% FCS. Cells were obtained from C3H responder mice (with
stimulator C57BL/6), Lewis (LEW) rats (with Brown Norway, BN, as
stimulator), and individual human donors. Culture supernatants were
harvested at 60 hrs and tested for different cytokines using
previously described ELISA assays (mouse), or using CTLL-2 as
bioassay for IL-2 production from all responder cell sources
(Gorczynski, R. M., et al. 1998c. Immunology. 93: p. 221-229).
Results
Evaluation of a Number of Mabs for Staining of Cell Populations in
Fresh PBL or Spleen:
All Mabs tested in the experiments herein described were previously
screened as described in the Materials and Methods above, and
detected a molecule in Western gel of brain extracts with Molecular
Weight 42-45 Kd, and also stained CHO transduced by OX-2 encoding
viral vectors. Data in Table 3 show FACS analysis for these Mabs
using fresh cells. The data are summed over several independent
analyses, using a number of Mabs directed to rat, mouse or human
OX-2, for staining of cells harvested from fresh PBL or spleen
(adherent cells only were tested for the latter: these represented
some 5%-8% of the total cell population in all cases).
It is clear from Table 3 that PBL in all species tested contained
some 1.3%-2.5% OX-2.sup.+ cells by FACS analysis, and that spleen
adherent cells similarly contained 4%-8% OX-2.sup.+ cells. As
confirmation of the inventor's previous work, spleen adherent cells
taken from C3H mice or LEW rats treated 4 days earlier by portal
venous immunization with 20.times.10.sup.6 (or 50.times.10.sup.6
respectively) of C57BL/6 (or BN) bone marrow cells showed some
3.5-5 fold elevation in OX-2.sup.+ cells (see Table 3). Under these
conditions specific increases in survival of subsequent
allo-transplanted cells/tissue have been reported (Gorczynski, R.
M. et al. 1996a. Transplantation 62:1592-1600).
Ability of Anti-OX-2 Mabs to Modulate Cytokine Production in MLR in
vitro:
In a final study the issue of whether these Mabs can modify the
immune response (as assayed by cytokine production) of cells
stimulated in an allogeneic mixed leucocyte reaction (MLR) in vitro
was addressed. The inventor has previously shown that cells taken
from mice pretreated by portal allogeneic immunization produce
predominantly type-2 cytokines, and that an anti-OX-2 Mab could
apparently reverse this polarization in cytokine production (and
indeed abolish the increased graft survival seen in such mice).
Data in Table 4 confirm these results using 3 independent Mabs to
mouse OX-2. Further, rat or human cells stimulated in the presence
of anti-rat (or human) OX-2, similarly show more pronounced IL-2
production than cells stimulated in the presence of isotypic
control Ig (or no Ig), without a generalized increase in cytokine
production (as analysed here by no change in IL-6 production in any
group).
Discussion
In the data in this example it is confirmed that using species
specific Mabs, to human, rat or mouse OX-2, that Mabs to the
molecule detected on the surface of host dendritic cells may play a
role in regulating cytokine production after allostimulation in
vitro, and more particularly that functionally blocking OX-2
expression leads to enhanced IL-2 production (a type-1 cytokine)
after allostimulation (Table 4). Borriello et al also recently
reported that OX-2 expression was not a costimulator for induction
of IL-2 and IFN.gamma. synthesis (Borriello, F. et al. 1997. J.
Immuno. 158:4548)-our data imply it is in fact a negative signal
for type-1 cytokine production. In mice preimmunized by the portal
vein, as reported earlier, there is a 4-fold increase in OX-2
expressing cells in PBL and spleen, and a reversal of polarization
in cytokine production (from type-2 cytokines to type-1 cytokines)
after stimulation of cells in the presence of OX-2 (see Tables 3
and 4) (Gorczynski, R. M. et al. 1998b. Transplantation.
65:1106-1114).
Example 5
Preparation of Rat Antibodies
Five rats were immunized using GERBU adjuvant (GERBU Biotechnik,
Gaiberg, Germany) with 500 mg of membrane protein purified from the
mouse dendritic cell (DC) line DC2.4 (a gift from K. Rock,
Harvard). Serum from these rats was tested 7 days after the third
immunization, and compared with a pre-immunization sample in an
ELISA using plate-bound material of Mol. Wt. 40 Kd-45 Kd eluted
from Western blots, and Alk Pase anti-rat Ig. Two rats with high
titre antibody were re-immunized and sacrificed 4 days later for
fusion of spleen cells with HAT-sensitive Sp2/0 parent cells for
preparation of hybridomas. Hybridomas were screened by ELISA
(56/960+ve), subcloned, and frozen (-70.degree. C.). For further
specificity testing of the anti-OX-2 Mabs will use CHO cells can be
transfected with a pBK eukaryotic expression vector (Stratagene,
Calif.) expressing OX-2. Full length OX-2 cDNA, including the
leader sequence, was amplified from DC2.4 cells using sense and
antisense primers constructed with Spe1 or Xba1 sites respectively
at their 5' ends for directional cloning into the vector. A band of
the expected size (849 bp) was obtained on agarose gel
electrophoresis. The sequence of the cloned cDNA was confirmed by
sequencing using an automated DNA sequencer (Chen, Z. and
Gorczynski, R. M. 1997. Biochem. Biophys. Acta. 100, in press). CHO
cells were transfected by electroporation (5.times.106 cells in 0.5
ml were pulsed at 960 MH.sub.2 and 120V using a Bio-Rad Gene Pulser
(Bio-Rad, Hercules, Calif.), using the full length OX-2 expression
plasmid along with a plasmid encoding puromycin resistance (100:1
ratio), followed by selection in puromycin (12 mg/ml for 4 days).
Puromycin resistant cells were cloned by limiting dilution. 5 CHO
transfectant clones have been obtained expressing mRNA for OX-2 as
confirmed by PCR. These clones can be used to screen the putative
rat anti-mouse OX-2 Mabs.
(a) FACS Staining of Cells from pv Immunized Mice with Anti-mouse
OX-2
A 4-fold increase in staining of spleen and hepatic NLDC145+
(dendritic cell marker) cells from pv immunized mice with anti-rat
OX-290 was observed. Spleen and liver tissue of mice at various
times (12 hours; 2, 7 and 14 days) following pv immunization can be
sectioned and stained by immunohistochemistry, using anti-NLDC145,
anti-OX-2 Mabs. Single cell suspensions from the same tissues can
be stained, using 3-colour FACS, with FITC-anti-mouse OX-2,
rhodamine-anti-NLDC145, and phycoerythrin-anti-T200 (mouse
lymphocyte marker). In all cases (both FACS and
immunohistochemistry) the appropriate irrelevant isotype control
antibodies are included. Tissue from control mice receiving renal
grafts alone, or following additional iv immunization, can also be
examined. Detection of NLDC145+ (and/or MAC-1+) cells showing
increased expression of OX-2 is predicted in pv immunized mice only
(see Gorczynski, R. M. et al. 1998. J. Immunol. 160, in press). The
inventor has shown DC-associated antigen persists only in animals
with surviving grafts (Gorczynski, R. M., Chen, Z., Zeng, H. and
Fu, X. M. 1998. Transplantation submitted). It was also assessed
whether anti-OX-2, infused at different times post transplantation,
causes rejection (b).
(b) Modulation of Graft Rejection and Cytokine Production by
Anti-mouse OX-2
C3H mice receive pv immunization with cultured C57BL/6 bone-marrow
derived dendritic cells (DC), CsA and renal allografts. Groups of
mice receive intravenous infusion of various rat anti-mouse OX-2
Mabs (100-500 mg/mouse, .times.5, at 2 day intervals), beginning at
different times post transplantation (this will be guided by data
from (a)). Serum creatinine and animal survival are followed. Serum
from Mab-treated mice are tested in ELISA and by FACS with OX-2
expressing CHO transfectants (above) to ensure antibody excess. If
OX-2 expression is important for pv induced increased graft
survival, the anti-OX-2 treated pv immunized mice will reject
grafts like untreated controls, with similar polarization of
cytokine production to type-1 cytokines (assayed by PCR; ELISA with
cultured, restimulated cells). As controls pv immunized, grafted
mice receive anti-CD28 and anti-CTLA4 these Mabs do not modify the
effects of pv immunization as assayed by graft survival or
polarization in cytokine production. It is expected that OX-2
treatment but not other Mabs, will simultaneously abolish expansion
of .gamma..delta.TCR+ cells after pv immunization.
Example 6
Preparation of a Fusion Protein Linking the Extracellular Domain of
OX-2 to Mouse Fc
Immunoadhesins, in which a hybrid molecule is created at the cDNA
level by fusing the extracellular domain (ED) of an adhesion
molecule with the carboxyl terminus of IgG heavy chain, the whole
being expressed in mammalian cells or in a baculovirus system, have
been powerful tools in the identification and isolation of the
counter ligands for the adhesion molecule of interest. Ligands for
a number of members of the TNFR family, were identified in this
fashion (Goodwin, R. G. et al. 1993. Eur. J. Immunol. 23,
2631-2641; Gruss, H. and Dower, S. 1995. Blood 85, 3378-3404).
Interest has developed in the potential application of
immunoadhesins as therapeutic agents. A CTLA4 immunoadhesion, with
the capacity to bind both B7-1 and B7-2, has been used to inhibit T
cell costimulation and decrease rejection (Larsen, C. P. et al.
1996. Nature 381, 434-438). Note that CD28/CTLA4 are not counter
ligands for OX-289. The fusion protein, is predicted to alter
cytokine production (increased IL-4, IL-10; decreased IL-2,
IFN.gamma.) and increase renal graft survival like pv immunization.
We expect that synergistic blockade of costimulation (e.g. by
CTLA4-Fc) and triggering of a coregulatory pathway (by OX-2ED-Fc)
will induce tolerance and produce indefinite graft survival.
a) Construction of an OX-2 Fusion Protein with Murine IgGFc2a
A cDNA encoding the extracellular region of OX-2 (OX-2ED) was
amplified by PCR, using a 5' oligonucleotide primer which inserts a
Sal1 site 5' immediately at the start of the V-region sequence and
a 3' primer which creates a BamH1 site at the 3' end (the site of
junction with Fc). Using cDNA prepared from mouse ConA activated
spleen cells, with a 5'primer containing an Spe1 site, and a 3'
primer containing a Sal1 site, the signal peptide for IL-6
(SP-IL-6) was amplified by PCR and ligated to the OX-2ED amplicon.
In frame ligation across the junction of SP-IL-6 and OX-2ED was
checked by manual sequencing-the final cDNA amplified by the
5'SP-IL-6 primer and the 3'OX-2ED primer was, as expected, 695bp. A
plasmid expressing murine IgGFc2a (Fcg2a), modified to create a
unique BamH1 site spanning the first codon of the hinge region, and
with a unique Xbal site 3' to the termination codon, has been
obtained from Dr. Terry Strom (Zheng, X. X. et al. 1995. Journal of
Immunology. 154, 5590-5600). The IgGFc2a in this insert has been
further modified to replace the C1q binding motif (rendering it
non-lytic) and inactivate the FcgRl binding site (see Zheng, X. X.
et al. 1995. Journal of Immunology. 154, 5590-5600). Ligation of
OX-2ED and IgGFc2a in the correct reading frame at the BamH1 site
yields a 1446 bp long open reading frame encoding a single
478-amino acid polypeptide (including the 24-amino acid IL-6 signal
peptide). The homodimer has a predicted 105 kDa Mol Wt, exclusive
of glycosylation. The fusion gene is then cloned as an Spe1-Xba1
cassette into the eukaryotic expression plasmid pBK/CMV
(Stratagene, Calif.). This plasmid has a CMV promoter/enhancer and
a neomycin-resistance gene for selection using G418. The
appropriate genetic construction of the OX-2ED-Fc can be confirmed
by direct sequencing after cloning into the plasmid vector (Chen,
Z. and Gorczynski, R. M. 1997. Biochem. Biophys. Acta. 100, in
press)--see also above. The plasmid is transfected into CHO cells
by electroporation (see above), and selected in medium with 1.5
mg/ml G418 (Geneticin:Life Technologies, Inc.). After subcloning,
high producing clones are selected by screening culture
supernatants in ELISA using anti-OX-2 Mabs as capture antibody, and
Alk Pase coupled anti-IgGFc2a as detection antibody. OX-2ED-Fc
fusion protein is purified from culture supernatants using protein
A-Sepharose affinity chromatography, dialysed against PBS,
filter-sterilized and stored in aliquots at -20.degree. C. The
size, and OX-2 (+IgGFc2a) specificity of the secreted product can
be confirmed using Western blot analysis under reducing (+DTT) and
non-reducing (-DTT) conditions, with Mabs to OX-2 and rat
monoclonal anti-mouse IgGFc2a (Pharmingen). The product can be
titrated as an inhibitor for FACS staining of OX-2 expressing CHO
cells (see above) using rat Mabs to OX-2 as probe. As a prelude to
studies (below) using OX-2ED-Fc in vivo, the half-life (t1/2) in
mouse serum following injection of groups of 6 8-week C3H mice will
be studied. This is carried out by subjecting mice to iv injections
of 50 mg or 10 mg of OX-2ED-Fc, and obtains serial 50 ml blood
samples at 0.3, 1, 6, 24, 48, 72 and 96 hours. The serum is
analyzed in ELISA using plates coated with anti-OX-2 as capture
antibody, and Alk Pase coupled monoclonal anti-IgGFc2a for
detection (thus ensuring the assay detects only OX-2ED-Fc, not OX-2
or IgGFc2a alone). Based on earlier data in which Fc fusion
proteins were used to extend the in vivo half-life, a t1/2 in the
range of 30-40 hrs (Zheng, X. X. et al. 1995. Journal of
Immunology. 154, 5590-5600) is predicted.
b) OX-2: IgGFc Immunoadhesion Inhibits MLR
CHO cells were transduced with a vector carry the OX-2:Fc cDNA
insert. Supernatant was harvested from the CHO cells at 7 days and
was cultured with 5.times.10.sup.6 LEW spleen and
2.5.times.10.sup.6 irradiated LBNFI spleen cells. The supernatant
contained 50 ng/ml OX-2:Fc.
The results, shown in Table 5, demonstrate that the soluble OX-2:Fc
immunoadhesion inhibits IL-2 production and generation of cytotoxic
T cells and induces IL-4 production. These results support the use
of OX-2 as an immunosuppressant.
c) Use of OX-2:Fc in vivo for Prevention of Graft Relection
It was shown in (b) that incubation in the presence of 50 ng/ml
OX-2:Fc can inhibit an in vitro MLR reaction. To detect inhibition
of in vivo graft rejection, C3H mice received C57BL/6 skin grafts
along with iv injection of OX-2:Fc (50 mg/mouse) every 2 days x4
injections. Grafts were inspected daily after 10 days for
rejection. In a separate study 3 mice/group (receiving saline or
OX-2:Fc) were sacrificed at 10 days and spleen cells restimulated
in vitro (.times.48 hrs) for analysis of cytokine production. Data
for these studies is shown in Tables 6 and 7. It is clear from
these data that OX-2:Fc has the potential for use as an
immunosuppressant to prolong graft acceptance. Furthermore, in
association with increased graft survival in this model, OX-2:Fc
alters polarization in cytokine production, as already described
for portal vein donor-specific immunization.
Example 7
OX-2 Expression in Placenta
Using in situ hybridization, the inventor has shown that OX-2 is
not expressed in the placenta of mice with increased potential for
fetal loss. In contrast, OX-2 is expressed in the placenta of
normal, non-aborting mice.
CBA/J and DBA/2J mice were used. Matings of CBA/J(females) with
DBA/2J males show a high incidence of fetal loss (>80%), unlike
the reverse scenario. Placental tissue was obtained from matings at
8-11 days of gestation. Uteri were snap frozen, 5 mm sections cut,
and stained with a biotinylated anti-sense probe for murine OX-2.
Data shown in FIGS. 18A and 18B indicate increased expression of
OX-2 mRNA (in situ labeling) in the non-aborting strain
combination, with essentially absent expression in the aborting
combination. These data are consistent with the notion that OX-2
expression prevents spontaneous fetal loss syndrome.
The data show that there are fewer OX-2+ implantation sites on day
8.5 of pregnancy in mice which are predisposed to fetal loss
syndrome (CBAxDBA/2 matings) by contrast to CBAxBALB/c matings
which are not so predisposed. FgI2 is the trigger for low, and
where OX-2 is also expressed, these potentially doomed
implantations are "rescued". This follows from the finding that the
abortion rate is lower than expected from % fgI2++ implantation
sites, unless anti-OX-2 mAb is administered. In the latter
instance, the abortion rate rises to equate with the estimated
proportion of fIg2++ implant sites.
Example 8
Materials and Methods
Mice:
Male C3H/HeJ, BALB/c and C57BL/6 mice were purchased from the
Jackson laboratories, Bar Harbour, Maine. Mice were housed 5/cage
and allowed food and water ad libitum. All mice were used at 8-12
weeks of age.
Monoclonal Antibodies: The following monoclonal antibodies (mAbs)
were obtained from Pharmingen (San Diego, Calif., USA) unless
stated otherwise: anti-IL-2 (S4B6, ATCC; biotinylated JES6-5H4);
anti-IL-4 (11B11, ATCC; biotinylated BVD6-24G2); anti-IFN.gamma.
(R4-6A2, ATCC; biotinylated XMG1.2); anti-IL-10 (JES5-2A5;
biotinylated, SXC-1); anti-IL-6 (MP5-20F3; biotinylated MP5-32C11);
anti-TNF.alpha. (G281-2626; biotinylated MP6-XT3); FITC anti-CD80,
FITC anti-CD86 and FITC anti-CD40 were obtained from Cedarlane
Labs, Hornby, Ontario. The hybridoma producing DEC205 (anti-mouse
dendritic cells) was a kind gift from Dr. R. Steinman, and was
directly labeled with FITC. FITC anti-H2K.sup.b, FITC
anti-H2K.sup.k, and anti-thy1.2 monoclonal antibodies (mAbs) were
obtained from Cedarlane Labs, Hornby, Ontario. Unconjugated and
PE-conjugated rat anti-mouse CD200 was obtained from BioSpark Inc.,
Mississauga, Ontario, Canada (Ragheb et al. 1999). CD200Fc was
prepared in a Baculovirus expression system, using a cDNA encoding
a murine IgG2aFc region (a kind gift from Dr. T Strom, Harvard,
USA) which carried mutations to delete complement binding and FcR
sites, as we described elsewhere (Gorczynski et al. 1999). Rat
monoclonal antibody to CD200.sup.r was prepared from rats immunized
with CHO cells transfected to express a cDNA encoding CD200.sup.r
(Gorczynski 2001). Anti-CD4 (GK1.5, rat IgG2b) and anti-CD8 (2.43,
rat IgG2b) were both obtained from ATCC, and used for in vivo
depletion by iv infusion of 100 .mu.g Ig/mouse weekly. A control
IgG2b antibody (R35.38), as well as strepavidin horse radish
peroxidase and recombinant mouse GM-CSF, was purchased from
Pharmingen (San Diego, Calif.).
Preparation of Cells: Single cell spleen suspensions were prepared
aseptically and after centrifugation cells were resuspended in
.alpha.-Minimal Essential Medium supplemented with
2-mercaptoethanol and 10% fetal calf serum (.alpha.F10).
CD200.sup.r+ LPS splenic Mph, stained (>20%) with FITC-CD200Fc,
were obtained by velocity sedimentation of cells cultured for 48
hrs with 1 mg/ml LPS (Gorczynski et al. 2000). Bone marrow cells
were flushed from the femurs of donor mice, washed and resuspended
in .alpha.F10. Cells were depleted of mature T lymphocytes using
anti-thy1.2 and rabbit complement.
C1498 (a spontaneous myeloid tumor) and EL4 (a radiation induced
thymoma tumor) cells were obtained from The American Type Culture
Collection (ATCC, Rockville, Md.). Cells used for transplantation
into mice were passaged weekly (5.times.10.sup.6 cells/mouse)
intraperitoneally in stock 8-week old C57BL/6 recipients. For
experimental tumor challenge either 5.times.10.sup.6 EL4 tumor
cells, or 5.times.10.sup.5 C1498 cells, were given
intraperitoneally to groups of 6 mice (see results)-animals were
sacrificed when they became moribund. EL4 cells stably transfected
to express CD80 or CD86 were obtained from Dr. J. Allison, Cancer
Research Labs, UC Berkeley, Calif., while C1498 transfected with
CD80/CD86 (cloned into pBK vectors) were produced in the author's
laboratory. Tumor cells (parent and transfected) were stored at
-80.degree. C. and thawed and cultured prior to use. Cells used for
immunization, including the tumor cells transfected with CD80/CD86,
were maintained in culture in aMEM medium supplemented with 10%
FCS. Untransfected and transfected cells of each tumor line were
used for immunization within 2 passages in culture. Over this time
in culture transfected cells repeatedly showed stable expression
(by FACS) of CD80/CD86 (>80% positive for each tumor assayed
over a 6 month period with multiple vials thawed and cultured).
Non-transfected tumor cells did not stain with these mAbs
(<2%).
CD200.sup.r+ cells were obtained from lymphocyte-depleted murine
spleen cells. Cells were treated with rabbit anti-mouse lymphocyte
serum and complement (both obtained from Cedarlane Labs. Hornby,
Ontario), cultured with LPS (10 .mu.g/ml) for 24 hours, and
separated into populations of different size by velocity
sedimentation (Gorczynski et al. 2000). Small CD200.sup.r+ cells
stained >65% by FACS with anti-CD200.sup.r antibody (Gorczynski
2001).
Bone Marrow Transplantation (BMT): C57BL/6 mice received 300 mg/Kg
cyclophosphamide iv 24 hrs before intravenous infusion of
20.times.10.sup.6 T-depleted C3H or C57BL/6 bone marrow cells.
Immediately prior to use for tumor transplantation (28 days
following bone marrow engrafting), a sample of PBL (50 .mu.l/mouse)
was obtained from the tail vein of individual mice and analysed by
FACS with FlTC-anti-H2K.sup.k or FlTC-anti-H2K.sup.b mAb. Cells
from normal C57BL/6 or C57BL/6 reconsituted C57BL/6 mice were 100%
H2K.sup.b positive, as expected. In similar fashion, PBL from C3H
mice were 100% H2K.sup.k positive. H2K.sup.k positive cells in the
C3H-reconstituted C57BL/6 mice by FACS comprised 85%.+-.8.5% of the
total cell population (mean over .about.100 mice used in the
studies described below). Mice in all groups were gaining weight
and healthy.
Cytotoxicity and Cytokine Assays:
In allogeneic mixed leukocyte cultures (MLC) used to assess
cytokine production or CTL, responder spleen cells were stimulated
with equal numbers of mitomycin-C treated (45 min at 37.degree. C.)
spleen stimulator cells in triplicate in .alpha.F10. Supernatants
were pooled at 40 hr from replicate wells and assayed in triplicate
in ELISA assays for lymphokine production as follows, using capture
and biotinylated detection mAbs as described above. Varying volumes
of supernatant were bound in triplicate at 4.degree. C. to plates
pre-coated with 100 ng/ml mAb, washed .times.3, and biotinylated
detection antibody added. After washing, plates were incubated with
strepavidin-horse radish peroxidase (Cedarlane Labs), developed
with appropriate substrate and OD.sub.405 determined using an ELISA
plate reader. Recombinant cytokines for standardization were
obtained from Pharmingen (U.S.A.). All assays showed sensitivity in
the range 40 to 4000 pg/ml. CTL assays were performed at 5 days
using cells harvested from the same cultures (as used for cytokine
assays). Various effector:target ratios were used in 4 hr .sup.51
Cr release tests with 72 hr ConA activated spleen cell blasts of
stimulator genotype.
Quantitation of CD200 mRNA by PCR:
RNA extraction from spleen tissue of tumor injected mice was
performed using Trizol reagent. The OD280/260 of each sample was
measured and reverse transcription performed using oligo (dT)
primers (27-7858: Pharmacia, USA). cDNA was diluted to a total
volume of 100 .mu.l with water and frozen at -70.degree. C. until
use in PCR reactions with primers for mouse CD200 and GAPDH
(Gorczynski et al. 1998). Different amounts of standard cDNA from
24 hr cultures of LPS stimulated peritoneal macrophages (known to
express CD200 and GAPDH) were amplified in six serial 1:10
dilutions for 30 cycles by PCR, in the presence of a tracer amount
of .sup.32 P. Samples were analysed in 12.5% polyacrylamide gels,
the amplicons cut from the gel, and radioactivity measured in a
.beta.-counter. A standard curve was drawn for each set of primer
pairs (amplicons). cDNAs from the various experimental groups were
assayed in similar reactions using 0.1 .mu.l cDNA, and all groups
were normalized to equivalent amounts of GAPDH. CD200 cDNA levels
in the different experimental groups were then expressed relative
to the CDNA standard (giving a detectable .sup.32 P signal over
five log.sub.10 dilutions). Thus a value of 5 (serial dilutions)
indicates a test sample with approximately the same cDNA content as
the standard, while a value of 0 indicates a test sample giving no
detectable signal in an undiluted form (<1/10.sup.5 the cDNA
concentration of the standard).
Results
Growth of EL4 or C1498 Tumor Cells in C57BU6 Mice, and in
Allogeneic (C3H) BMT Mice:
Groups of 6 C57BL/6 mice received iv infusion of 300 mg/Kg
cyclophosphamide (in 0.5 ml PBS). A control group received PBS
only, as did a control group of 6 C3H mice. 24 hrs later
cyclophosphamide treated C57BL/6 mice received iv injection of
20.times.10.sup.6 T-depleted bone marrow cells pooled from C57BL/6
mice (syngeneic transplant), or C3H mice (allogeneic
transplantation). All groups of animals received intraperitoneal
injection (in 0.5 ml PBS) of 5.times.10.sup.6 EL4 or
5.times.10.sup.5 C1498 tumor cells (see FIG. 19) 28 days later.
Animals were monitored daily post tumor inoculation.
Data in FIG. 19 (one of 2 such studies) show clearly that while C3H
mice rejected both EL4 and C1498 (allogeneic) leukemia cell growth,
100% mortality was seen within 9-12 days in normal C57BL/6 mice, or
in syngeneic bone marrow reconstituted mice. Interestingly, despite
the absence of overt GVHD (as defined by weight loss and overall
health), two-thirds of C3H reconstituted C57BL/6 mice rejected EL4
tumor cells, reflecting the existence of a graft versus leukemia
effect (GVL) (panel a of FIG. 19), and there was a marked delay of
death for mice inoculated with C1498 leukemia cells (panel b of
Figure). In separate studies similar findings were made using tumor
inocula (for EL4/C1498 respectively) ranging from 2.times.10.sup.6
-10.times.10.sup.6, or 1.5.times.10.sup.5 -1.times.10.sup.5
(RMG-unpublished).
Immunization of Normal C57BL/6 Mice for Protection Against EL4
Tumor Growth:
Blazar and co-workers reported immunization for protection from
tumor growth in C57BL/6 mice using tumor cells transfected to
over-express mouse CD80 (Blazar et al. 1997). Using CD80 and CD86
transfected EL4 cells obtained from this same group, or C1498 cells
tranfected with CD80/CD86 in our laboratory, we immunized groups of
6 C57BL/6 mice ip with Complete Freund's adjuvant (CFA) alone, or
with CFA mixed with 5.times.10.sup.6 mitomycin-C treated tumor
cells, or CD80/CD86 transfected tumor. Animals received 2
injections at 14 day intervals. 10 days after the last immunization
all mice received 5.times.10.sup.6 EL4 tumor cells, or
5.times.10.sup.5 C1498 cells, and mortality followed. Data are
shown in FIG. 20 (1 of 2 studies), for EL4 only.
In agreement with a number of other reports, mice pre-immunized
with CD80-transfected EL4 survive significantly longer after
challenge with viable EL4 tumor cells than non-immunized animals,
or those immunized with non-transfected cells or CD86 transfected
cells (p<0.05)--see also FIG. 23. Similar data were obtained
using CD80-transfected C1498 cells (RMG-unpublished). In separate
studies (not shown) mice immunized with tumor cells in the absence
of Freund's Adjuvant failed to show any protection from tumor
growth. However, equivalent protection (to that seen using Freund's
Adjuvant) was also seen using concomitant immunization with
poly(I:C) (100 .mu.g/mouse) as adjuvant (data not shown).
Role of CD4.sup.+ and/or CD8.sup.+ Cells in Modulation of Tumor
Growth after BMT:
In order to investigate the effector cells responsible for leukemia
growth-inhibition in mice transplanted with allogeneic bone marrow
(see FIG. 19), BMT recipients received weekly injections of 100
.mu.g/mouse anti-CD4 (GK1.5) or anti-CD8 (2.43) mAb, followed at 28
days by leukemia cell injection as described for FIG. 19. Depletion
of CD4 and CD8 cells in all mice with these treatments was >98%
as defined by FACS analysis (not shown).
As shown in FIG. 21 (data from one of 3 studies), and in agreement
with data reported elsewhere (Blazar et al. 1997), in this BMT
model tumor growth inhibition for EL4 cells is predominantly a
function of CD8 rather than CD4 cells, while for C1498 leukemia
cells growth inhibition was equally, but not completely, inhibited
by infusion of either anti-CD4 or anti-CD8 mAb (see pane b of
Figure).
Evidence that Tumor Relection in BMT Mice is Regulated by
CD200:
Previous studies in rodent transplant models have implicated
expression of a novel molecule, CD200, in the regulation of an
immune rejection response. Specifically, blocking functional
expression of CD200 by a monoclonal antibody to murine CD200
prevented the increased graft survival which followed
donor-specific pretransplant immunization, while a soluble form of
CD200 linked to murine IgG Fc (CD200Fc) was a potent
immunosuppressant (Gorczynski et al. 1998; Gorczynski et al. 1999).
In order to investigate whether expression of CD200 was involved in
regulation of tumor immunity, we studied first the effect of
infusion of CD200Fc on suppression of resistance to growth of EL4
or C1498 tumor in BMT mice as described in FIG. 19, and second the
effect of CD200Fc infusion in mice immunized with CD80-transfected
tumor cells as described in FIG. 20. Note this CD200Fc lacks
binding sites for mouse complement and FcR (see Materials and
Methods, and Gorczynski et al. 1999). In all cases control groups
of mice received infusion of equivalent amounts of pooled normal
mouse IgG. Data for these studies is shown in FIGS. 22 and 23
respectively (data from one of 2 studies in each case).
It is clear that suppression of growth of either EL4 or C1498 tumor
cells in BMT mice is inhibited by infusion of CD200Fc, but not by
pooled normal mouse IgG (FIG. 22). CD200Fc also caused increased
mortality in EL4 or C1498 injected normal C3H mice. Data in FIG. 23
show that resistance to EL4 tumor growth in EL4-CD80 immunized mice
(as documented in FIG. 20) is also inhibited by infusion of
CD200Fc. In separate studies (not shown) a similar inhibition of
immunity induced by CD80 transfected C1498 was demonstrated using
CD200Fc.
Effect of Anti-CD200 mAb on Resistance to Tumor Growth in Mice
Immunized with CD80ICD86 Transfected Tumor Cells:
As further evidence for a role for CD200 expression in tumor
immunity in EL4-CD80/EL4-CD86, or C1498-CD80/C1498-CD86 immunized
mice we examined the effect of infusion of an anti-CD200 mAb on EL4
or C1498 tumor growth in this model. Infusion of anti-CD200 into
mice preimmunized with EL4-CD86, or C1498-CD86 uncovered evidence
for resistance to tumor growth. Separate studies (not shown)
revealed that anti-CD200 produced no significant perturbation of
EL4 growth in the EL4 or EL4-CD80 immunized mice, or of C1498
growth in C1498 or C1498-CD86 immunized mice. We interpreted these
data to suggest that immunization with EL4-CD86 or C1498-CD86
elicited an antagonism of tumor immunity resulting from increased
expression of CD200. Thus blocking the functional increase of CD200
expression with anti-CD200 reversed the inhibitory effect. Note
that in studies not shown we have reproduced these same effects of
anti-CD200 (in mice immunized with CD86-transfected tumor cells)
with F(ab').sub.2 anti-CD200 (RMG-unpublished).
To confirm that indeed immunization with CD86-transfected tumor
cells was associated with increased expression of CD200, we
repeated the study shown in FIG. 24, and sacrificed 3 mice/group at
4 days following EL4 tumor injection. RNA was isolated from the
spleen of all mice, and assayed by quantitative PCR for expression
of CD200 (using GAPDH as "housekeeping" mRNA control). Data for
this study are shown in FIG. 25, and show convincingly that CD200
mRNA expression was >5-fold increased following preimmunization
with EL4-CD86, a condition associated with increased tumor growth
compared with EL4-immunized mice (FIGS. 20 and 24). In
dual-staining FACS studies (not shown), with PE-anti-CD200 and
FITC-DEC205, the predominant CD200.sup.+ population seen in control
and immunized mice were DEC205+ (>80%)--see also Gorczynski et
al. (1999). Similar results were obtained using C1498 tumor cells
(data not shown).
Given this increase in CD200 expression following preimmunization
with CD86-transfected cells, and the evidence that CD200 is
associated with delivery of an immunosuppressive signal to antigen
encountered at the same time, we also examined the response of
spleen cells taken from these C57BL/6 mice to allostimulation (with
mitomycin-C treated BALB/c spleen cells), in the presence/absence
of anti-CD200 mAb. Data from one of 3 studies are shown in Table 8.
Interestingly, mice preimmunized with EL4-CD86 cells show a
decreased ability to generate CTL on alloimmunization with
third-party antigen (BALB/c), and decreased type-1 cytokine
production (IL-2, IFN.gamma.), with some trend to increased type-2
cytokines (IL-4 and IL-10). These effects were reversed by
inclusion of anti-CD200 in culture, consistent with the hypothesis
that they result from increased delivery of an immunosuppressive
signal via CD200 in spleen cells obtained from these animals
(Gorczynski et al. 1998).
Evidence for an Interaction Between CD200 and CD200.sup.r+ Cells in
Inhibition of EL4 Tumor Growth:
Inhibition resulting from infusion of CD200Fc into mice follows an
interaction with immunosuppressive CD200.sup.r+ cells (Gorczynski
et al. 2000). At least one identifiable functionally active
population of suppressive CD200.sup.r+ cells was described as a
small, F4/80.sup.+ cell in a pool of splenic cells following LPS
stimulation (Gorczynski et al. 2000)--F4/80 is a known cell surface
marker for tissue macrophages. In a further study we investigated
whether signaling induced by CD200:CD200.sup.r interaction (where
CD200.sup.r+ cells were from lymphocyte-depleted, LPS stimulated,
spleen cells) was behind the suppression of tumor immunity seen
following CD200Fc injection. All groups of 6 recipient mice
received tumor cells ip. In addition to infusion of CD200Fc as
immunosuppressant (in FIG. 26), one group of C3H reconstituted
animals received CD200.sup.r+ cells (in FIG. 26: >65% of these
cells stained with an anti-CD200.sup.r mAb), while a final group
received a mixture of both CD200Fc and CD200.sup.r+ cells. It is
clear from the Figure that it is this final group, in which
interaction between CD200 and CD200.sup.r is possible, which showed
maximum inhibition of tumor immunity compared with the C3H
reconstituted control mice.
Role of CD4.sup.+ and/or CD8.sup.+ Cells in CD200 Regulated
Modulation of Tumor Growth after BMT:
Data in FIG. 21 above, and elsewhere (Blazar et al. 1997) show that
tumor growth inhibition for EL4 cells is predominantly a function
of CD8 rather than CD4 cells, while for C1498 leukemia cells growth
inhibition was equally, but not completely, inhibited by infusion
of either anti-CD4 or anti-CD8 mAb. To investigate the role of
CD200 in the protection mediated by different T cell subclasses,
the following additional studies were performed. In the first,
C57BL/6 recipients of C3H BMT received (at 28 days post BMT)
inoculations of EL4 or C1498 tumor cells along with anti-CD4,
anti-CD8 or CD200Fc alone, or combinations of (anti-CD4+CD200Fc) or
(anti-CD8+CD200Fc). Survival was followed as before (see FIG.
27--one of 2 studies). In a second study (FIG. 28--data from one of
2 such experiments) a similar treatment regimen of mAbs or CD200Fc
alone or in combination was used to modify growth of EL4 tumor
cells in mice preimmunized with EL4-CD80 cells as described earlier
in FIG. 20.
Data in panel a of FIG. 27 confirm the effects previously
documented in FIGS. 21 and 22, that CD200Fc and anti-CD8 each
significantly impaired the growth inhibition in BMT recipients of
EL4 cells, while anti-CD4 mAb was less effective. Combinations of
CD200Fc and either anti-T cell mAb led to even more pronounced
inhibition of tumor immunity in the BMT recipients, to levels seen
with non-allogeneic transplanted mice. Data with C1498 tumor cells
(panel b) were somewhat analogous, though as in FIG. 21, anti-CD4
alone produced equivalent suppression of growth inhibition to
anti-CD8 with this tumor. As was the case for the EL4 tumor,
combinations of CD200Fc and either anti-T cell mAb caused
essentially complete suppression of C1498 tumor growth inhibition.
Both sets of data, from panels a and b, are consistent with the
notion that CD200Fc blocks (residual) growth-inhibitory functional
activity in both CD4 and CD8 cells, thus further inhibiting tumor
immunity remaining after depletion of T cell subsets.
Data in FIG. 28, using EL4 immunized BL/6 mice, also showed
combinations of CD200Fc and anti-CD4 treatment produced optimal
suppression of tumor immunity to EL4 cells, consistent with an
effect of CD200Fc on CD8.sup.+ cells. Anti-CD8 alone abolished
tumor immunity in these studies, so any potential additional
effects of CD200Fc on CD4.sup.+ cells could not be evaluated.
Discussion
In the studies described above, we have asked whether expression of
the molecule CD200, previously reported to down-regulate rejection
of tissue/organ allografts in rodents (see previous Examples), was
implicated in immunity to tumor cells in syngeneic hosts. Two model
systems were used. The one, in which tumor cells are injected into
mice which had received an allogeneic bone marrow transplant
following cyclophosphamide pre-conditioning, has been favoured as a
model for studying potential innovative treatments of
leukemia/lymphoma in man (Blazar et al. 1997; Imamura et al. 1996;
Blazar et al. 1999; Champlin et al.1999). In the other EL4 or C1498
tumor cells were infused into BL/6 mice which had been preimmunized
with tumor cells transfected to overexpress the costimulatory
molecules CD80 or CD86. These studies were stimulated by the
growing interest in such therapy for immunization of human tumor
patients with autologous transfected tumor cells (Imro et al. 1998;
Brady et al. 2000; Jung et al. 1999; Freund et al. 2000;
MartinFontecha et al. 2000).
In both sets of models we found evidence for inhibition of tumor
growth (FIGS. 19 and 20) which could be further modified by
treatment designed to regulate expression of CD200. Infusion of
CD200Fc suppressed tumor immunity (led to increased tumor growth,
and faster mortality) in both models (FIGS. 22 and 23), while
anti-CD200 improved tumor immunity in mice immunized with
CD86-transfected EL4 or C1498 tumor cells (FIG. 24). We interpreted
this latter finding as suggesting that the failure to control tumor
growth following immunization with EL4-CD86 or C1498-CD86 was
associated with overexpression of endogenous CD200, a hypothesis
which was confirmed by quantitative PCR analysis of tissue taken
from such mice (FIG. 25). CD200 was predominantly expressed on
DEC205.sup.+ cells in the spleen of these mice (see text), which
was associated with a decreased ability of these spleen cell
populations to respond to allostimulation in vitro (see Table 8).
Non antigen-specific inhibition following CD200 expression formed
the basis of our previous reports that a soluble form of CD200
(CD200Fc) was a potent immunosuppressant (Gorczynski et al. 1998).
Consistent with the hypothesis that increased expression of CD200
in mice immunized with CD86-transfected tumor cells was responsible
for the inhibition of alloreactivity seen in Table 8, suppression
was abolished by addition of anti-CD200 mAb (see lower half of
Table 8). Earlier reports have already documented an
immunosuppressive effect of CD200Fc on alloimmune responses
(Gorczynski et al. 1999), and production of antibody in mice
following immunization with sheep erythrocytes (Gorczynski et al.
1999).
Maximum inhibition of tumor immunity was achieved by concomitant
infusion of CD200Fc and CD200r+ cells (F4/80.sup.+ macrophages--see
FIG. 26). We next investigated the cell type responsible for tumor
growth inhibition whose activity was regulated by CD200:CD200.sup.r
interactions. Our data confirmed previous reports that EL4 growth
inhibition was predominantly associated with CD8 immune cells,
while immunity to C1498 was a function of both CD4.sup.+ and
CD8.sup.+ cells (Blazar et al. 1997). For both tumors in BMT models
suppression of tumor growth inhibition was maximal following
combined treatment with CD200Fc and either anti-T cell mAb,
consistent with the idea that CD200 suppression acts on both
CD4.sup.+ and CD8.sup.+ T cells.
A number of studies have examined immunity to EL4 or C1498 tumor
cells in similar models to those described above (Blazar et al.
1997; Boyer et al. 1995), concluding that CD8.sup.+ cells are
important in (syngeneic) immunity to each tumor, and CD4.sup.+ T
cells are also important in immunity to C1498 (Blazar et al. 1997).
Evidence to date suggests that NK cell mediated killing is not
relevant to tumor growth inhibition in BMT mice of the type used
above (Blazar et al. 1997). Other reports have addressed the issue
of the relative efficiency of induction of tumor immunity in a
number of models following transfection with CD80 or CD86, and also
concluded that CD80 may be superior in induction of anti-tumor
immunity (Blazar et al. 1997; Chen et al. 1994), while CD86 may
lead to preferential induction of type-2 cytokines (Freeman et al.
1995). This is of interest given the cytokine production profile
seen in EL4-CD86 immunized mice (Table 8), which is similar to the
profile seen following CD200Fc treatment of allografted mice
(Gorczynski et al. 1998). EL4-CD86 immunized mice show increased
expression of CD200 (FIG. 25), with no evidence for increased
resistance to tumor growth (FIG. 20). Resistance is seen in these
mice following treatment with anti-CD200 (FIG. 24A). Somewhat
better protection from tumor growth is seen using viable tumor
cells for immunization, rather than mitomycin-C treated cells as
above (Blazar et al. 1997). Whether this would improve the degree
of protection from tumor growth in our model, and/or significantly
alter the role of CD200:CD200.sup.r interactions in its regulation,
remains to be seen.
There are few studies exploring the manner in which suppression
mediated by CD200:CD200.sup.r interactions occurs. In a recent
study in CD200 KO mice Hoek et al observed a profound increase in
the presence of activated macrophages and/or macrophage-like cells
(Hoek et al. 2000), and we and others had previously found that
CD200 r was expressed on macrophages (Gorczynski et al. 2000;
Wright et al. 2000). The inventors also reported that CD200.sup.r
was present on a subpopulation of T cells, including the majority
of activated .gamma..delta.TCR.sup.+ cells (Gorczynski et al.
2000), a result we have recently confirmed by cloning a cDNA for
CD200.sup.r from such cells (Kai et al--in preparation).
.gamma..delta.TCR+cells may mediate their suppressive function via
cytokine production (Gorczynski et al. 1996), while unpublished
data (RMG-in preparation) suggests that the CD200.sup.r+ macrophage
cell population may exert its activity via mechanisms involving the
indoleamine 2,3-dioxygenase (IDO) tryptophan catabolism pathway
(Mellor et al. 1999). We suggest that the mechanism by which
CD200Fc leads to suppression of tumor growth inhibition in the
models described is likely to be a function both of the tumor
effector cell population involved (FIGS. 27, 28) as well as the
CD200.sup.r cell population implicated in suppression.
In a limited series of studies (not shown) we have used other BMT
combinations (B10 congenic mice repopulated with B10.D2, B10.BR or
B10.A bone marrow) to show a similar resistance to growth of EL4 or
C1498 tumor cells, which is abolished by infusion of CD200Fc.
Studies are in progress to examine whether DBA/2 or BALB/c mice can
be immunized to resist growth of P815 syngeneic (H2.sup.d) tumor
cells by P815 cells tranfected with CD80/CD86, and whether this too
can be abolished by CD200Fc. Taken together, however, our data are
consistent with the hypothesis that the immunomodulation following
CD200:CD200.sup.r interactions, described initially in a murine
allograft model system, is important also in rodent models of tumor
immunity. This has important implications clinically.
Example 9
TRIM in the FSL Sarcoma Lung Metastasis Model: Cues from
Pregnancy
Allogeneic leukocyte-induced transfusion-related immunomodulation
(TRIM) has been shown to enhance tumor growth (Vamvakas et al.
1994; Bordin et al. 1994). OX-2 is expressed on a variety of cells
in transfused blood (i.e. a subpopulation of dendritic cells and
possibly B cells) (Wright et al. 2000; Hoek et al. 2000), thus the
effect of anti-OX-2 on the TRIM enhancement of FSL10 lung nodules
was examined.
Materials and Methods
Enhancement of Lung Nodules by TRIM
A dose response curve demonstrated a plateau in the TRIM
enhancement of lung metastases with 50, 100 or 200 .mu.l of BALB/c
heparinized blood given 4 days after tail vein injection of the
cultured tumor cell by tail vein (see FIG. 29). A dose of 200 .mu.l
of BALB/c heparinized blood (about 15-20% of blood volume) was
given 4 days after tail vein injection of the cultured tumor cells
as a physiologically suitable model in which to screen for
treatments that have a major abrogating effect on TRIM.
All animals were monitored for signs of illness daily, and 21 days
after tumor inoculation, the mice were sacrificed, the lungs were
removed and fixed in Bouin's solution, and the number of surface
nodules was counted. To deal with variation in number of metastases
between mice, 20-25 mice per group were used and medians were
calculated (using log-transformed data, where 0 nodules was set at
0.1 for that animal). It was then possible to assess the
significance of differences in log mean .+-.sem with respect to our
a priori hypotheses using Student's t test, and to construct 95%
confidence intervals for the medians. Differences in the proportion
of mice in different groups with no visible metastases was assessed
by the .chi..sup.2 statistic, or by Fisher's Exact test where
appropriate.
FIG. 29A shows the median number of lung nodules in C57BI/6J mice
receiving the indicated dose of freshly-prepared allogeneic BALB/c
strain blood by tail vein. The effect is seen if the blood is given
7 days prior to, or 4 days after a tail vein injection of
1.times.10 6 FSL sarcoma cells. FSL10 is a
methlycholanthrene-induced fibrosarcoma generated in C57BI/6 mice
and maintained by standard tissue culture in vitro. Such cells are
weakly antigenic. Group size is 20-25 per group, and P values
showing increased numbers to lung nodules are on the figure. FIG.
29B shows the proportion of mice with no tumor nodules. P values
were determined by Student's t test for A, and by Chi-square or
Fisher's Exact test for B.
The Role of Dendritic Cells
MAb to a myeloid DC/APC surface marker (5 .mu.g anti-CD11c) or
lymphoid dendritic cells (DC) (5 .mu.g supernatant of DEC205
hybridoma, an amount shown to be sufficient using in vitro assays
of DC function (Gorczynski et al. 2000)) was added to 200 .mu.l of
BALB/c blood or to PBS. The TRIM enhancement of tumor growth was
analyzed using the same method as above.
Results
Enhancement of Lung Nodules by TRIM
FIG. 30A represents the effect of adding anti-OX-2 monoclonal
antibody (3B6, 1 ug per million leukocytes) to the blood (or PBS
control) before tumor cell transfer. A control is the same amount
of 3B6 in PBS. The total dose was 3.3 ug per mouse. FIG. 30A shows
this amount of anti-CD200 in PBS had no effect, whereas when added
to blood, the stimulation of tumor nodule number was
prevented,--indeed, it was reduced below control levels. FIG. 30B
shows % with no lung nodules. In this and subsequent studies,
2.times.10 6 FSL cells were used, and the blood was always given 4
days after this.
As illustrated in FIG. 30, the enhancement of lung nodules in mice
given 2.times.10.sup.5 sarcoma cells by 200 .mu.l of BALB/c blood
compared to phosphate buffered saline control (PBS) given 4 days
after tumor injection, was completely blocked by adding 3.3 .mu.g
of anti-OX-2 (3B6 monoclonal antibody (mAb)) to the blood before
the transfusion. The mAb in PBS had no effect. Interestingly, the
proportion of mice with lung metastases was boosted by allogeneic
blood compared to PBS but was reduced by blood to which anti-OX-2
had been added. The median number of nodules was greater in this
study in part because we had doubled the tumor cell inoculum, but
we do see experiment-to-experiment variation in the number of
nodules in the control group which has been important in executing
large experiments, as will be discussed.
The Role of Dendritic Cells
FIG. 31A is a repetition of FIG. 30A which confirms the effect of
anti-OX-2, but with addition of antibodies to dentritic cells.
Anti-CD11c was used for myeloid dendritic cells, and DEC205 for
lymphoid dendritic cells. The latter are usually CD8-positive. It
can be readily appreciated that monoclonal antibody to lymphoid
dendritic cells had no effect on the stimulation of lung
metastases, whereas anti-CD11c blocked the effect. The reason the
number of nodules is not below control is thought to be due to the
existence of OX-2-positive and OX-2-negative CD11c-type dendritic
cells. The latter stimulate immunity, and this is seen when
anti-OX-2 is used to block one of the subsets. Anti-CD11c leads to
loss of both subsets.
The results show that anit-CD11c, but not DEC205, abrogated the
TRIM effect (* indicates significant increase over control, **
indicates significant abrogation of TRIM, *** indicates significant
decrease below control, P<0.05). There was no effect of
anti-OX-2, DEC205 or anti-CD11c in PBS injected as a control (data
not shown). Due to the large number of treatment groups in this
experiment, it could not be done in a single day. Therefore, 5 mice
in each group were treated in 4 experiments and the data was
examined and pooled. The result therefore compensates for any
effect of day-to-day variation in tumor cells, mice or blood used
for transfusion.
While the present invention has been described with reference to
what are presently considered to be the preferred examples, it is
to be understood that the invention is not limited to the disclosed
examples. To the contrary, the invention is intended to cover
various modifications and equivalent arrangements included within
the spirit and scope of the appended claims.
All publications, patents and patent applications are herein
incorporated by reference in their entirety to the same extent as
if each individual publication, patent or patent application was
specifically and individually indicated to be incorporated by
reference in its entirety.
TABLE 1 Summary of sequences and clones detected in cDNA library
from pv immunized mice Match category Number of clones represented
(%) Known mouse genes 30 (45) Non-mouse genes (rat/human) 10 14
(21) No data base match 22 (34) Footnotes: Genes were considered a
"match" with a BLAST score >250 with a minimum of 50 bp
alignment.
TABLE 2 Cytokine production from cells of mice receiving pv
immunization and anti-rat OX-2 Cytokine levels in culture
supernatants.sup.b Mabs given to recipients.sup.a IL-2 IFN.gamma.
IL-4 IL-10 No pv immunization (CsA only) None 750 .+-. 125 85 .+-.
18 29 .+-. 8 130 .+-. 40 +anti-rat OX-2 890 .+-. 160 93 .+-. 19 30
.+-. 10 120 .+-. 35 +anti-mouse CD28 415 .+-. 88* 57 .+-. 9* 105
.+-. 22* 275 .+-. 55* +anti-mouseCTLA4 505 .+-. 125* 65 .+-. 8 95
.+-. 20* 190 .+-. 45 +anti-B7-1 340 .+-. 65* 35 .+-. 7* 120 .+-.
21* 285 .+-. 60* +anti-B7-2 495 .+-. 90* 64 .+-. 7 90 .+-. 20* 185
.+-. 45 PV Immunization + CsA None 190 .+-. 55 25 .+-. 8 107 .+-.
21 780 .+-. 150 +anti-rat OX-2 730 .+-. 140* 60 .+-. 16* 33 .+-.
10* 220 .+-. 40* +anti-mouse CD28 145 .+-. 38 20 .+-. 9 145 .+-. 34
1140 .+-. 245 +anti-mouseCTLA4 85 .+-. 25 15 .+-. 6 125 .+-. 31 960
.+-. 220 +anti-B7-1 110 .+-. 30 20 .+-. 6 144 .+-. 28 885 .+-. 180
+anti-B7-2 75 .+-. 20 14 .+-. 5 150 .+-. 30 1230 .+-. 245
Footnotes: .sup.a 3 C3H mice/group were used in each experiment.
All animals received CsA and C57BL/6 renal transplants as described
in the Materials and Methods. Mice in the lower half of the Table
also received pv infusions of 15 .times. 10.sup.6 C57BL/6 bone
marrow derived dendritic cells on the day of transplantation. Where
monoclonal antibodies were given the dose used was 100 mg/mouse,
.times.4 doses at 2 day intervals. # All mice were sacrificed 14
days post transplantation. Spleen cells were culture in triplicate
from individual animals for 40 hrs in a 1:1 mixture with irradiated
C57BL/6 spleen stimulator cells. .sup.b Arithmetic mean (.+-.SD)
for triplicate determinations from individual samples of the
animals described in the first column. All cytokines were assayed
by ELISA. IL-2, IL-4 and IL-10 are shown as pg/ml, IFN.gamma. as
ng/ml. Data are pooled from 2 such studies (total of 6 individual
mice tested/group). *represents significantly different from
control group with no Mab (p <0.02)
TABLE 3 FACS staining of PBL and spleen adherent cells in different
species, using anti-OX-2 Mabs Donor.sup.b Percent stained
cells.sup.c SPECIES.sup.a Treatment Mab PBL Spleen Human NONE H4B4
1.5 .+-. 0.3 4.8 .+-. 1.7 H4A9A2 1.5 .+-. 0.4 6.1 .+-. 2.0 H4A9C7
1.3 .+-. 0.4 4.3 .+-. 1.7 Mouse NONE M3B5 1.9 .+-. 0.4 6.7 .+-. 2.1
M3B6 1.7 .+-. 0.4 5.2 .+-. 1.6 M2C8 1.4 .+-. 0.4 4.2 .+-. 1.4 Mouse
PV immune M3B5 5.9 .+-. 1.5 20 .+-. 4.1 M3B6 5.2 .+-. 1.4 17 .+-.
3.6 M2C8 4.7 .+-. 1.4 15 .+-. 3.3 Rat NONE RC6A3 1.3 .+-. 0.3 5.3
.+-. 1.6 RC6C2 1.5 .+-. 0.4 6.5 .+-. 1.7 RC6D1 1.9 .+-. 0.6 6.8
.+-. 1.5 Rat PV immune RC6A3 4.8 .+-. 1.3 16 .+-. 4.2 RC6C2 4.9
.+-. 1.6 18 .+-. 3.9 RC6D1 5.3 .+-. 1.7 20 .+-. 4.5 Footnotes:
.sup.a Fresh cells were obtained from normal human donors (PBL),
cadaveric transplant donors (human spleen), or from adult (8-10
week) mouse or rat donors. The same 3 separate tissue donors were
used for each Mab tested. .sup.b Donor pretreatment refers to
infusion of allogeneic bone marrow cells into the portal vein
(C57BL/6 for C3H mouse donors; BN for LEW rat donors) 4 days before
harvest of PBL or spleen (see text and (6)). .sup.c Arithmetic mean
(+SD) for percent cells stained in 3 independent assays. Control
antibodies (FITC anti-mouse IgG (for anti-human or anti-rat Mabs,
or FITC anti-rat IgG for anti-mouse Mabs) gave no significant
staining above background (<0.2%).
TABLE 4 Type-1 cytokine production in MLR cultures is increased by
anti-OX-2 Mabs Cytokine levels in culture supernatants.sup.b ELISA
assays (murine only) Bioassay (CTTL-2) Mabs in culture.sup.a IL-2
IFN.sub..gamma. IL-4 IL-10 IL-2 IL-6 MOUSE MLR None 350 .+-. 55 35
.+-. 18 345 .+-. 63 340 .+-. 50 480 .+-. 160 365 .+-. 74 M3B5 890
.+-. 160* 115 .+-. 29* 130 .+-. 10* 168 .+-. 42* 820 .+-. 200* 265
.+-. 46 M3B6 915 .+-. 155* 117 .+-. 25* 135 .+-. 32* 135 .+-. 38*
850 .+-. 175* 303 .+-. 55 M2C8 855 .+-. 155* 105 .+-. 28* 120 .+-.
32* 140 .+-. 37* 830 .+-. 165* 279 .+-. 61 control Ig 370 .+-. 75
36 .+-. 11 330 .+-. 55 310 .+-. 45 335 .+-. 60 349 .+-. 59 None**
710 .+-. 145 108 .+-. 23 110 .+-. 21 105 .+-. 23 690 .+-. 155 285
.+-. 54 RAT MLR None 490 .+-. 145 360 .+-. 57 RC6A3 690 .+-. 155*
295 .+-. 55 RC6C2 845 .+-. 180* 345 .+-. 68 RC6D1 830 .+-. 160* 370
.+-. 57 Control Ig 475 .+-. 160 356 .+-. 58 HUMAN MLR None 395 .+-.
85 295 .+-. 45 H4B4 570 .+-. 125* 315 .+-. 50 H4A9A2 630 .+-. 145*
320 .+-. 48 H4A9C7 625 .+-. 140* 345 .+-. 56 Control Ig 360 .+-.
120 320 .+-. 50 Footnotes: .sup.a MLR cultures were set up as
described in the Materials and Methods. For human MLR cultures the
same 3 different responder preparations were used for each Mab, and
stimulated with a pool of mitomycin C treated spleen stimulator
cells (from a random mixture of 6 spleen donors). For mouse (C3H
anti-C57BL/6) and rat (LEW anti-BN) MLR cultures all assays were
set up in triplicate for each Mab. Mouse responder spleen cells
were from mice treated 4 # days earlier by portal vein infusion of
C57BL/6 bone marrow cells, except for data shown as (None**) where
responder cells were from non-injected C3H mice. Mab was added as a
30% superntatant concentration. Supernatants were harvested for
cytokine assays at 60 hrs. .sup.b Data show arithmetic means (+SD)
for each Mab. For mouse assays all supernatants were assayed for a
number of cytokines (ELISA), and for IL-2/IL-6 using bioassays
(proliferation of CTLL-2, B9 respectively). Supernatants from
rat/human cultures were assayed in bioassays only. Note that cells
incubated with isotype control Igs (non-reactive by ELISA or FACS)
gave cytokine data indistinguishable from cultures incubated in the
absence of # Mab. p < 0.05, compared with cultures without
Mabs.
TABLE 5 OX-2:FC Immunoadhesin Inhibits Mixed Leukocyte Reaction in
vitro Percent lysis Added supernatant.sup.a .sup.51 Cr
targets.sup.b Cytokines in culture (pg/ml).sup.c (50:1,
effector:target) IL-2 IL-4 NONE (control) 31 .+-. 4.0 1005 .+-. 185
60 .+-. 20 Control CHO 33 .+-. 4.3 810 .+-. 190 45 .+-. 20 (vector
transduced) CHO transduced with 4.2 .+-. 2.1 175 .+-. 45 245 .+-.
55 OX-2:Fc Footnotes: .sup.a Supernatant was harvested at 7 days
from CHO cells transduced with control pbK vector, or vector
carrying a cDNA insert encoding OX-2 linked to murine Fc. A 1:1
mixture of supernatant was used in cultures containing 5 .times.
10.sup.6 LEW spleen and 2.5 .times. 10.sup.6 irradiated LBNF1
spleen cells; this corresponded to 50 ng/ml OX-2:Fc .sup.b
and.sup.c Percent lysis with cells at 5 days, using 1 .times.
10.sup.4 51 Cr BN spleen ConA targets; cytokines in culture
supernatants at 60 hrs.
TABLE 6 Inhibition of skin graft rejection by OX-2:Fc Rejection of
skin grafts Treatment of mice (mean + SD) in days NIL 12 + 3.8
OX-2:Fc 19 + 4.2 Footnotes: 6 mice/group were treated as shown. NIL
indicates infusion of normal mouse IgG only. Arithmetic mean (+SD)
graft survival for group.
TABLE 7 OX-2:Fc infused into mice receiving skin allografts
reverses polarization in cytokine production Cytokines in
supernatant at Treatment of mice culture 48 hrs (pg/ml) IL-2 IL-4
NIL 1250 + 160 80 + 20 OX-2:Fc 350 + 85 245 + 50 Footnotes: 3
mice/group received iv infusions of saline or OX-2:Fc (50 mg/mouse)
every 2 days .times.4 from the time of grafting with C57BL/6 skin.
Mice were sacrificed at 10 days and spleen cells stimulated in
vitro with irradiated C57BL/6 spleen stimulator cells. Arithmetic
mean (+SD) for IL-2/IL-4 in supernatant at 48 hrs. Data are pooled
from triplicate cultures for each mouse spleen.
TABLE 8 Preimmunization of mice with EL4-CD86 causes increased
CD200 expression which leads to generalized suppression to newly
encountered alloantigen Cytokines in Supernatant.sup.c Tumor used
for Immunization.sup.a % Lysis.sup.b IL-2 IL-4 IFN.sub..gamma.
IL-10 NONE (control) 43 .+-. 5.5 980 .+-. 125 50 .+-. 10 455 .+-.
65 35 .+-. 10 EL4 41 .+-. 6.2 890 .+-. 135 60 .+-. 15 515 .+-. 70
30 .+-. 10 EL4-CD80 46 .+-. 6.3 955 .+-. 140 45 .+-. 15 525 .+-. 55
30 .+-. 10 EL4-CD86 16 .+-. 4.2* 420 .+-. 75* 125 .+-. 20* 240 .+-.
40* 120 .+-. 20* NONE (control) + 46 .+-. 5.8 950 .+-. 105 55 .+-.
15 490 .+-. 60 40 .+-. 10 EL4 + 44 .+-. 4.9 940 .+-. 115 50 .+-. 20
530 .+-. 60 35 .+-. 10 EL4-CD80 + 44 .+-. 6.0 905 .+-. 120 60 .+-.
15 555 .+-. 75 35 .+-. 10 EL4-CD86 + 39 .+-. 4.29 870 .+-. 125 75
.+-. 20 540 .+-. 65 40 .+-. 15 Footnotes: .sup.a Spleen cells were
pooled from 3 C57BL/6 mice/group, pretreated as described in the
text, by immunization with 5 .times. 10.sup.6 mitomycin-C treated
EL4 tumor cells, or CD80/CD86-transfected tumor cells, in Complete
Freund's Adjuvant 4 days earlier. 5 .times. 10.sup.6 spleen cells
were incubated in triplicate with equal numbers of mitomycin-C
treated BALB/c spleen stimulator cells. + indicates anti-CD200
(anti-OX2) added to cultures (5 .mu.g/ml) .sup.b % specific lysis
in 4-hr .sup.51 Cr release assays with 72-hr cultured BALB/c spleen
Con A blast cells (effector:target ratio shown is 100:1). .sup.c
Cytokines in culture supernatants assayed in triplicate by ELISA at
40 hrs (see Materials and Methods). Data represent pg/ml except for
IL-10 (ng/ml). *p < 0.05 compared with all groups.
Full Citations for References Referred To in the Specification
Ahvazi, B. C., P. Jacobs, and M. M. Stevenson. 1995. Role of
macrophage-derived nitric oxide in suppression of lymphocyte
proliferation during blood-stage malaria. J. Leu. Biol. 58
(1):23-31. Akatsuka, Y., C. Cerveny, and J. A. Hansen. 1996. T cell
receptor clonal diversity following allogeneic marrow grafting.
Hum. Immunol. 48:125-134. Akolkar, P. N., B. Gulwani-Akolkar, R.
Pergolizzi, R. D. Bigler, and J. Silver. 1993. Influence of HLA
genes on T cell receptor V segment frequencies and expression
levels in peripheral blood lymphocytes. J. Immunol. 150 (April
1):2761-2773. Albina, J. E., J. A. Abate, and W. L. Henry. 1991.
Nitric oxide production is required for murine resident peritoneal
macrophages to suppress mitogen-stimulated T cell proliferation. J.
Immunol. 147:144-152. Banchereau, J., and R. M. Steinman. 1998.
Dendritic cells and the control of immunity. Nature. 392:245-252.
Barclay, A. N. 1981. Different reticular elements in rat lymphoid
tissue identified by localization of la, Thy-1 and MRC OX-2
antigens. Immunology 44:727 Barclay, A. N., and H. A. Ward. 1982.
Purification and chemical characterization of membrane
glycoproteins from rat thymocytes and brain, recognized by
monoclonal antibody MRC OX-2. Eur. J. Biochem. 129:447. Blazar B R,
Taylor P A, Boyer M W, PanoskaltsisMortari A, Allison J P, Vallera
D A: CD28/B7 interactions are required for sustaining the
graft-versus-leukemia effect of delayed post-bone marrow
transplantation splenocyte infusion in murine recipients of myeloid
or lymphoid leukemia cells. J Immunol, 1997; 159:3460-3473. Blazar,
B. R., Taylor P. A., PanoskaltsisMorari, A., Sharpe, A. H. and
Vabera, D. A. 1999 J. Immunol 162:6368. Bordin, J. O., Bardossy,
L., and Blajchman, M. A. 1994 Growth enhancement of established
tumors by allogeneic blood transfusion in experimental animals and
its amelioration by leukodepletion: the importance of timing of the
leukodepletion. Blood 84:344. Borriello, F., J. Lederer, S. Scott,
and A. H. Sharpe. 1997. MRC OX-2 defines a novel T cell
costimulatory pathway. J. Immuno. 158:4548. Boyer M W, Orchard P J,
Gorden K, Andersen P M, Mcivor R S, Blazar B R: Dependency upon
intercellular adhesion molecule (ICAM) recognition and local IL-2
provision in generation of an in vivo CD8+ T cell immune response
to murine myeloid leukemia. Blood, 1995; 85:2498-2505 Brady, M. S.,
Lee, F., Eckels, D. D., Ree, S. Y., Latouche, J. B. and Lee, J. S.
2000 J Immunother 23:353-361. Brasel, K., H. J. McKenna, K.
Charrier, P. J. Morrissey, D. E. Williams, and S. D. Lyman. 1997.
Flt3 ligand synergizes with granulocyte-macrophage
colony-stimulating factor or granulocyte colony-stimulating factor
to mobilize hematopoietic progenitor cells into the peripheral
blood of mice. Blood. 90:3781-3788. Bronstein, I., J. C. Voyta, O.
J. Murphy, L. Bresnick, and L. J. Kricka. 1992. Improved
chemiluminescence Western blotting procedure. Biotechniques 12:748.
Castle, B. E., K. Kishimoto, C. Stearns, M. L. Brown, and M. R.
Kehry. 1993. Regulation of the expression of the ligand for CD40 on
T helper lymphocytes. J. Immunol. 151:1777. Champlin R., Khouri,
I., Kornblau, S., Marini, F., Anderlini, P., Ueno, N., Molldrem, J.
and Giralt, S. 1999 Hematol Oncol Clin N Amer 13:1041. Chen L,
McGowan P, Ashe S, Johnston J, Li Y, Hellstrom K E, Hellstrom K E:
Tumor immunogenicity determines the effect of B7 costimulation on
T-cell mediated tumor immunity. J Exp Med, 1994; 179:523-530. Chen,
Z., H. Zeng, and R. M. Gorczynski. 1997. Cloning and
characterization of the murine homologue of the rat/human MRC OX-2
gene. BBA. Mol. Basis Dis. 1362:6-10. Chen, Z. and Gorczynski, R.
M. 1997. Biochem. Biophys. Acta. 100, in press. Clark, D. A., J.-W.
Ding, G. Yu, G. A. Levy and R. Gorczynski. 2001. Fgl2
prothrombinase expression in mouse trophoblast and decidua triggers
abortion but may be counted by OX-2. Mol. Human Reproduction, Vol.
7, No. 2, pp.185-194. Freeman, G. J., V. A. Boussiotis, A.
Anumanthan, G. M. Bernstein, X. Y. Ke, P. D. Rennert, G. S. Gray,
J. G. Gribben, and L. M. Nadler. 1995. B7-1 and B7-2 do not deliver
identical costimulatory signals, since B7-2 but not B7-1
preferentially costimulates the initial production of IL-4.
Immunity. 2:523-532. Freund Y R, Mirsalis J C, Fairchild D G, Brune
J, Hokama L A, SchindlerHorvat J, Tomaszewski J E, Hodge J W,
Schlom J, Kantor J A, Tyson C A, Donohue S J: Vaccination with a
recombinant vaccinia vaccine containing the B7-1 co-stimulatory
molecule causes no significant toxicity and enhances T
cell-mediated cytotoxicity. Int J Cancer, 2000; 85:508-517.
Garside, P., and A. M. Mowat. 1997. Mechanisms of oral tolerance.
Crit. Rev. Immunol. 17:119-137. Goodwin, R. G., Din, W. S.,
Davis-Smith, T. et al. 1993. Eur. J. Immunol. 23, 2631-2641
Gorczynski, R. M. 1992. Immunosuppression induced by hepatic portal
venous immunization spares reactivity in IL-4 producing T
lymphocytes. Immunol. Lett. 33:67-77. Gorczynski, R. M., and D.
Wojcik. 1992. Antigen presentation by murine splenic, but not
hepatic, antigen-presenting cells to induce IL-2/IL-4 production
from immune T cells is regulated by interactions between
LFA-1/ICAM-1. Immunol. Lett. 34:177-182. Gorczynski, R. M., and D.
Wojcik. 1994. A role for non-specific (cyclosporin A) or specific
(monoclonal antibodies to ICAM-1, LFA-1 and interleukin-10)
immunomodulation in the prolongation of skin allografts after
antigen-specific pre-transplant immunization or transfusion. J.
Immunol. 152:2011-2019. Gorczynski, R. M., Z. Chen, S. Chung, Z.
Cohen, G. Levy, B. Sullivan, and X.-M. Fu. 1994a. Prolongation of
rat small bowel or renal allograft survival by pretransplant
transfusion and/or by varying the route of allograft venous
drainage. Transplantation 58:816-820. Gorczynski, R. M. 1994b.
Adoptive transfer of unresponsiveness to allogenic skin grafts with
hepatic .gamma..delta.+ T cells. Immunology 81:27-35. Gorczynski,
R. M. 1995a. Regulation of IFN.gamma. and IL-10 synthesis in vivo,
as well as continuous antigen exposure, is associated with
tolerance to murine skin allografts. Cell. Immunol. 160:224-231.
Gorczynski, R. M., N. Hozumi, S. W. Wolfe, and Z. Chen. 1995b.
Interleukin-12, in combination with anti-interleukin-10, reverses
graft prolongation after portal venous immunization.
Transplantation. 60:1337-1341. Gorczynski, R. M., Z. Cohen, X. M.
Fu, Z. Hua, Y. L. Sun, and Z. Q. Chen. 1996a. Interleukin-13, in
combination with anti interleukin-12, increases graft prolongation
after portal venous immunization with cultured allogenic bone
marrow-derived dendritic cells. Transplantation 62:1592-1600.
Gorczynski, R. M., Z. Cohen, G. Levy, and X. M. Fu. 1996b. A role
for gamma delta TCR(+) cells in regulation of rejection of small
intestinal allografts in rats. Transplantation. 62:844-851.
Gorczynski R M, Cohen Z, Leung Y, Chen Z: gamma delta TCR(+)
hybridomas derived from mice preimmunized via the portal vein
adoptively transfer increased skin allograft survival in vivo. J
Immunol, 1996; 157:574-581. Gorczynski, R. M., Z. Chen, Y. Hoang,
and B. RossiBergman. 1996c. A subset of gamma delta T-cell
receptor-positive cells produce T-helper type-2 cytokines and
regulate mouse skin graft rejection following portal venous
pretransplant preimmunization. Immunology. 87 (3):381-389.
Gorczynski, R. M., Z. Chen, H. Zeng, and X. M. Fu. 1998a. A role
for persisting antigen, antigen presentation and ICAM-1 in the
increased renal graft survival following oral or portal vein
donor-specific immunization. Transplantation. 66: 000-008.
Gorczynski, R. M., Z. Chen, X. M. Fu, and H. Zeng. 1998b. Increased
expression of the novel molecule Ox-2 is involved in prolongation
of murine renal allograft survival. Transplantation. 65:1106-1114.
Gorczynski, R. M., et al. 1998c. Analysis of cytokine production
and V beta T-cell receptor subsets in irradiated recipients
receiving portal or peripheral venous reconstitution with
allogeneic bone marrow cells, with or without additional
anti-cytokine monoclonal antibodies. Immunology. 93: p. 221-229.
Gorczynski, R. M., Chen, Z., Fu, X. M. and Zeng, H. 1998. J.
Immunol. 160, in press. Gorczynski, R. M., Chen, Z., Zeng, H. and
Fu, X. M. 1998. Transplantation submitted. Gorczynski, R. M., Chen,
Z., Fu, X. M., and Zeng, H. 1998. Increased expression of the novel
molecule OX-2 is involved in prolongation of murine renal allograft
survival. Transplantation 65:1106-1114. Gorczynski, R. M., Cattral,
M. S., Chen, Z. G., Hu, A., Lei, J., Min, W. P., Yu, G., and Ni, J.
1999 An immunoadhesion incorporating the molecule OX-2 is a potent
immunosuppressant that prolongs allo- and xenograft survival. J
Immunol 163:1654-1660. Gorczynski L, Chen Z, Hu J, Kai G,
Ramakrishna V, Gorczynski R M: Evidence that an OX-2 positive cell
can inhibit the stimulation of type-1 cytokine production by
bone-marrow-derived B7-1 (and B7-2) positive dendritic cells. J
Immunol, 1999; 162:774-781 Gorczynski, R. M., Yu, G., and Clark, D.
A. 2000 J. Immunol 166, submitted. Gorczynski, R. M., Chen, Z.,
Clark D. A., Hu, J., Yu G., Li, X., Tsang, W., and Hadidi, S. 2000
Regulation of gene expression of murine MD-1 regulates subsequent T
cell activation and cytokine production. J. Immuol. 165:1925.
Gorczynski R M, Yu K, Clark D: Receptor engagement on cells
expressing a ligand for the tolerance-inducing molecule OX2 induces
an immunoregulatory population that inhibits alloreactivity in
vitro and in vivo. J Immunol, 2000; 165:4854-4860. Gorczynski R M:
Transplant tolerance modifying antibody to CD200 receptor (CD200r),
but not CD200, alters cytokine production profile from stimulated
macrophages. Europ J Immunol, 2001; 100:001-006. Gruss, H. and
Dower, S. 1995. Blood 85, 3378-3404 Hancock, W. W., M. H. Sayegh,
X. G. Zheng, R. Peach, P. S. Linsley, and L. A. Turka. 1996.
Costimulatory function and expression of CD40 ligand, CD80, and
CD86 in vascularized murine cardiac allograft rejection. Proc.
Natl. Acad. Sci. USA. 93:13967-13972. Hoek, R. M., Ruuls, S. R.,
Murphy, C. A., Wright G. J., Goddard, R., Zurawski, S. M., Blom,
B., Homola, M. E., Streit, W. J., Brown M. H., Barclay, A. N.,
Sedgwick J. D. 2000. Down-regulation of the macrophage lineage
through interaction with OX2 (CD200). Science 290:1768-1771.
Imamura M, Hashino S, Tanaka J: Graft-versus-leukemia effect and
its clinical implications. Leuk Lymphoma, 1996; 23:477-492 Imro, M.
A., Dellabona, P., Manici, S., Heltai, S., Consogno, G., Bellone,
M., Rugarli, C., and Protti M. P. 1998 Human melanoma cells
transfected with the B7-2 co-stimulatory molecule induce
tumor-specific CD8(+) cytotoxic T lymphocytes in vitro. Human Gene
Ther 9:1335-1344. Jenkins, M. K., J. D. Ashwell, and R. H.
Schwartz. 1988. Allogeneic non-T spleen cells restore the
responsiveness of normal T cell clones stimulated with antigen and
chemically modified antigen-presenting cells. J. Immunol.
140:3324-3329. Jung D, Hilmes C, Knuth A, Jaeger E, Huber C,
Seliger B: Gene transfer of the co-stimulatory molecules B7-1 and
B7-2 enhances the immunogenicity of human renal cell carcinoma to a
different extent. Scand J Immunol, 1999; 50:242-249. Kenick, S., R.
P. Lowry, R. D. S. Forbes, and R. Lisbona. 1987. Prolonged cardiac
allograft survival following portal venous inoculation of
allogeneic cells: What is "hepatic tolerance?". Transpl. Proc.
19:478-480. Kohler, G. and C. Milstein. 1975. Preparation of
monoclonal antibodies. Nature. 25: p. 256-259. Kronin, V., K.
Winkel, G. Suss, A. Kelso, W. Heath, J. Kirberg, H. vonBoehmer, and
K. Shortman. 1996. Subclass of dendritic cells regulates the
response of naive CD8 T cells by limiting their IL-2 production. J.
Immunol. 157:3819-3827. Kuchroo, V. K., M. P. Das, J. A. Brown, A.
M. Ranger, S. S. Zamvil, A. Sobel, H. L. Weiner, N. Nabavi, and L.
H. Glimcher. 1995. B7-1 and B7-2 costimulatory molecules activate
differentially the Th1/Th2 developmental pathways: application to
autoimmune disease therapy. Cell. 80:707-718. Larsen, C. P., S. C.
Ritchie, R. Hendrix, P. S. Linsley, K. S. Hathcock, R. J. Hodes, R.
P. Lowry, and T. C. Pearson. 1994. Regulation of immunostimulatory
function and costimulatory molecule (B7-1 and B7-2) expression on
murine dendritic cells. J. Immunol. 152:5208-5219. Larsen, C. P.,
Elwood, E. T., Alexander, D. Z. et al. 1996. Nature 381, 434-438
Larsen, C. P., and T. C. Pearson. 1997. The CD40 pathway in
allograft rejection, acceptance, and tolerance. Curr. Opin.
Immunol. 9:641-647. Leenen, P. J. M., K. Radosevic, J. S. A.
Voerman, B. Salomon, N. vanRooijen, D. Klatzmann, and W. vanEwijk.
1998. Heterogeneity of mouse spleen dendritic cells: In vivo
phagocytic activity, expression of macrophage markers, and
subpopulation turnover. J. Immunol. 160:2166-2173. Lenschow, D. J.,
T. L. Walunas, and J. A. Bluestone. 1996. CD28/B7 system of T cell
costimulation. Annu. Rev. Immunol. 14:233-258. Maraskovsky, E., K.
Brasel, M. Teepe, E. R. Roux, S. D. Lyman, K. Shortman, and H. J.
McKenna. 1996. Dramatic increase in the numbers of functionally
mature dendritic cells in Fit 3 Ligand-treated mice: multiple
dendritic cell subpopulations identified. J. Exptl. Med.
184:1953-1962. MartinFontecha A, Moro M, Crosti M C, Veglia F,
Casorati G, Dellabona P: Vaccination with mouse mammary
adenocarcinoma cells coexpressing B7-1 (CD80) and B7-2 (CD86)
discloses the dominant effect of B7-1 in the induction of antitumor
immunity. J Immunol , 2000; 164:698-704 Mayer, L. 1996. Yin and
Yang of mucosal immunology. Transpl. Proc. 28:2435-2437. McCaughan,
G. W., et al. 1987. The gene for MRC OX-2 membrane glycoprotein is
localized on human chromosome 3. Immunogenetics. 25: p. 133-135.
Mellor A L, Munn D H: Tryptophan catabolism and T-cell tolerance:
immunosuppression by starvation? Immunol Today, 1999; 20:469-473.
Miller, R. G., and R. A. Phillips. 1969. The separation of cells by
velocity sedimentation. J. Cell. Comp. Physiol. 73:191-198.
Preston, S., et al. 1997. The leukocyte/neuron cell surface antigen
OX2 binds to a ligand on macrophages. Eur J Immunol. 27(8): p.
1911-8. Qian, J. H., T. Hashimoto, H. Fujiwara, and T. Hamaoka.
1985. Studies on the induction of tolerance to alloanatigens. I.
The abrogation of potentials for delayed-type hypersensitivity
responses to alloantigens by portal venous inoculation with
allogeneic cells. J. Immunol. 134:3656-3663. Ragheb, R., Abrahams,
S., Beecroft, R., Hu, J., Hi, J., Ramakrishna, V., Yu, G. and
Gorczynski, R. M. 1999. Preparation and functional properties of
monoclonal antibodies to human, mouse and rat OX-2. Immunology
Letters 68:311-315. Rosenzwajg, M., S. Camus, M. Guigon, and J. C.
Gluckman. 1998. The influence of interleukin (IL)-4, IL-13, and
Flt3 ligand on human dendritic cell differentiation from cord blood
CD34(+) progenitor cells. Exp. Hematol. 26:63-72. Salomon, B., J.
L. Cohen, C. Masurier, and D. Klatzmann. 1998. Three populations of
mouse lymph node dendritic cells with different origins and
dynamics. J. Immunol. 160:708-717. Sandhu, G. S., B. W. Eckloff,
and B. C. Kline. 1991. Chemiluminescent substrates increase
sensitivity of antigen detection in Western blots. Biotechniques
11:14. Schwartz, R. H. 1996. Models of T cell anergy: Is there a
common molecular mechanism? J Exp Med. 184: p. 1-8. Steinbrink, K.,
M. Wolfi, H. Jonuleit, J. Knop, and A. H. Enk. 1997. Induction of
tolerance by IL-10-treated dendritic cells. J Immunol.
159:4772-4780. Steptoe, R. J., F. M. Fu, W. Li, M. L. Drakes, L. A.
Lu, A. J. Demetris, S. G. Qian, H. J. McKenna, and A. W. Thomson.
1997. Augmentation of dendritic
cells in murine organ donors by Flt3 ligand alters the balance
between transplant tolerance and immunity. J Immunol.
159:5483-5491. Suss, G., and K. Shortman. 1996. A subclass of
dendritic cells kills CD4+ T cells via Fas/Fas-ligand-induced
aoptosis. J. Exptl. Med. 183:1789-1796. Swain, S. L. 1995. Who does
the polarizing? Curr. Biol. 5:849-851. Thelen, M., and U.
Wirthmueller. 1994. Phospholipases and protein kinases during
phagocytic activation. Curr. Opin. Immunol. 6:106-112. Vamvakas, E.
and Moore, S. B. 1994 Blood transfusion and postoperative septic
complications. Tranfusion 33:754. Wright, G. J., Puklavec, M. J.,
Willis, A. C., Hoek, R. M., Sedgwick J. D., Brown, M. H., Barclay,
A. N. 2000 Lymphoid/neuronal cell surface OX2 glycoprotein
recognizes a novel receptor on macrophages implicated in the
control of their function. Immunity 13:233-242. Zheng, X. X.,
Steele, A. W., Nickerson, P. W. et al. 1995. Journal of Immunology
154, 5590-5600.
SEQUENCE LISTING <100> GENERAL INFORMATION: <160>
NUMBER OF SEQ ID NOS: 22 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 1 <211> LENGTH: 2791 <212> TYPE:
DNA <213> ORGANISM: Mus musculus <400> SEQUENCE: 1
actatagggc acgcgtggtc gacggcccgg gctggtactg agaaggaata ggatgcagtc
60 agagggaagg gacttgagga agacctttgg tttagactct ctccacatgt
ctgtctgtgg 120 gtctctgaac cagattttat ctgttgctgc ctctctgatg
acagctggtc aaggccccaa 180 tctattagta tagcagaatg tcattaagaa
tcattctttt cttccttcca ttttttcttc 240 ttcttctact ccctccccct
ttctctctct ctctttcttt ctttctttct ttctttttct 300 ttctttcttt
ctctctctcc ctctttctct ctctccctct ctctctttct ttctttcttt 360
ctttctcttt ttctttctgt ctttctctct ctttccttct ttccttcccc agctgtgttt
420 ggttctaccc taggactctg ggctttccat tatctggttc ttgaccatcc
aggcaatata 480 ggtacaagct ctctcttata gggtgggcct caagttaaac
taaacattgg ttggtcactc 540 cctcacgttt tctactaaaa tctcataggc
aggacatatt gtgggtagag gatttagagg 600 caggtttagt gtccaggttt
gtcttttcat ggtctgtaga ataccttctc acaccagaga 660 gactagagtc
tagagtccaa acctcagctc tagcctctct atgttcagtg agctgaatga 720
aagttgacct cagcaatggg tcccactgtc aggttttaga gggtgacctt cagttgtagg
780 tcccaagtct ctctctcctc tctctccctt tctcgatctc tctctctctc
tctctctctc 840 tctctctctc tctctctctc tctctctctc tctctctgct
ttatacttgt gattgaagat 900 gtgatctctc tggcagcctg gtaccatgcc
tcctggtcac ttagagactc tcctcctgta 960 gctataagcc caacaaatct
ttttccacag gtttctactc tagtacagaa acagaaatgt 1020 caccaatata
gtcaatcgtt tctgtaaagc tttcatcaag gaaaacctca gttccagggc 1080
ttcctgtgac tcatttgatc tgtcccttga ttctcatctg ttttaaggaa tactgcggga
1140 caatctgatt agcagaaaga aagtgctttt gggttttcag gaagtgtgtt
cacaggtagc 1200 tctgagccct taggacttct aaagctctag atgaggtacc
tggtaaccac acacacacac 1260 acacacacac acacacacac acacgcactg
gcctttaata taacaaatca taaaataaag 1320 tttttctttt tttttcccca
gggtgtctgt atgaatctcc ttaccttctt ccccctacac 1380 acacacacac
acacacacac acacacacac acactattgt tctgttctcc gagtttacct 1440
tttgctgtac agaaccacag gatgcaccgg gtttctgact caaattactg tccactcaag
1500 ttagttccca ctccgatttt tctgtatgga ctacgtcacc ctatactgcc
atttggcacg 1560 ggagagaggc cagtgatggg aatgcagacg aaacatgcat
acacatgtaa aataagataa 1620 ataaatctaa aatgaaaaaa aatatagagt
gattctttca catttttgct atattactct 1680 aaaaggcgag aacctggcgg
gggcgggggc aggggctagg gacgaggttg tagagggcgt 1740 ggttggttgg
tcgtctcttc ctccacacta gaggagctgt agagtctgcc tgtgcggtgg 1800
agggggctct ctctacggcg aatagtagtg tccctgctca caggtgttgc ggagatatcc
1860 tccatcgtgg aagagctcag accccgagaa gctggtgtct agctgcggcc
ccgagcaagg 1920 atgggcagtc tggtgagtgg aatctgagat gcgaaggagg
gcggaatggg cgatctggag 1980 ccgcggctct cagaagccag tggagcctgc
gagaaaagca aggaagctgt tctttggaga 2040 agtggtatcc ggggctcgga
gctctgtaag gaggcaccgg ccggagaaag cccggggaac 2100 gcgtgtatct
agggtgggcg gctttgctcc ttgctgcgat tccattgcga aaacacggcc 2160
tgagctccat ggctcccaga aggggaggag tagctctttg cgtcccctat gttggtcctt
2220 aacctgcagc aggggtgtag cctagtaatc tcgcttgctc tctttctcac
cccctctctt 2280 gctgcatttc tgctccttgc ctagaaaacc atgaagcatc
tagcagtact gcagcgagca 2340 agccacagct tagtggtctt gttaaatgcc
aaggtattta gaggagaggc cgacattttg 2400 agtctttggt actgtttaca
aggcagaaaa ttttaaaagg aagggtggtc atacgcctta 2460 ttctttatac
acacggaatt ggtagaattg aatgcgaatc taaacgcaat taaaccccag 2520
gtaccacttt tcatcaggct gacaaagacc gacttgtgtt acctttccta acaaagagga
2580 atgtggatct gtcagctaga tgctcttagt gttcaaacaa ggaattgctt
tctgttttac 2640 aaagaatcgg agagagaggt tctttttttt ctctccaagt
ctctgtggct gcaatgaaat 2700 aaggtacaaa atcagaccta gaaagaatag
gggaatgggg ctatgcacct agcagaccag 2760 cccgggccgt cgaccacgcg
tgccctatag t 2791 <200> SEQUENCE CHARACTERISTICS: <210>
SEQ ID NO 2 <211> LENGTH: 278 <212> TYPE: PRT
<213> ORGANISM: Mus musculus <400> SEQUENCE: 2 Met Gly
Ser Leu Val Phe Arg Arg Pro Phe Cys His Leu Ser Thr Tyr 1 5 10 15
Ser Leu Ile Trp Gly Met Ala Ala Val Ala Leu Ser Thr Ala Gln Val 20
25 30 Glu Val Val Thr Gln Asp Glu Arg Lys Ala Leu His Thr Thr Ala
Ser 35 40 45 Leu Arg Cys Ser Leu Lys Thr Ser Gln Glu Pro Leu Ile
Val Thr Trp 50 55 60 Gln Lys Lys Lys Ala Val Ser Pro Glu Asn Met
Val Thr Tyr Ser Lys 65 70 75 80 Thr His Gly Val Val Ile Gln Pro Ala
Tyr Lys Asp Arg Ile Asn Val 85 90 95 Thr Glu Leu Gly Leu Trp Asn
Ser Ser Ile Thr Phe Trp Asn Thr Thr 100 105 110 Leu Glu Asp Glu Gly
Cys Tyr Met Cys Leu Phe Asn Thr Phe Gly Ser 115 120 125 Gln Lys Val
Ser Gly Thr Ala Cys Leu Thr Leu Tyr Val Gln Pro Ile 130 135 140 Val
His Leu His Tyr Asn Tyr Phe Glu Asp His Leu Asn Ile Thr Cys 145 150
155 160 Ser Ala Thr Ala Arg Pro Ala Pro Ala Ile Ser Trp Lys Gly Thr
Gly 165 170 175 Thr Gly Ile Glu Asn Ser Thr Glu Ser His Phe His Ser
Asn Gly Thr 180 185 190 Thr Ser Val Thr Ser Ile Leu Arg Val Lys Asp
Pro Lys Thr Gln Val 195 200 205 Gly Lys Glu Val Ile Cys Gln Val Leu
Tyr Leu Gly Asn Val Ile Asp 210 215 220 Tyr Lys Gln Ser Leu Asp Lys
Gly Phe Trp Phe Ser Val Pro Leu Leu 225 230 235 240 Leu Ser Ile Val
Ser Leu Val Ile Leu Leu Val Leu Ile Ser Ile Leu 245 250 255 Leu Tyr
Trp Lys Arg His Arg Asn Gln Glu Arg Gly Glu Ser Ser Gln 260 265 270
Gly Met Gln Arg Met Lys 275 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 3 <211> LENGTH: 14 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer <400> SEQUENCE: 3
ttttgtacaa gctt 14 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 4 <211> LENGTH: 44 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Adapter 1 <400> SEQUENCE: 4
ctaatacgac tcactatagg gctcgagcgg ccgcccgggc aggt 44 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 5 <211>
LENGTH: 43 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Adapter 2 <400> SEQUENCE: 5 tgtagcgtga agacgacaga aagggcgtgg
tgcggagggc ggt 43 <200> SEQUENCE CHARACTERISTICS: <210>
SEQ ID NO 6 <211> LENGTH: 22 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer 1 <400> SEQUENCE: 6
ctaatacgac tcactatagg gc 22 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 7 <211> LENGTH: 22 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Nested Primer 1 <400>
SEQUENCE: 7 tcgagcggcc gcccgggcag gt 22 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 8 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Primer 2
<400> SEQUENCE: 8 tgtagcgtga agacgacaga a 21 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 9 <211>
LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Nested
Primer 2 <400> SEQUENCE: 9 agggcgtggt gcggagggcg gt 22
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 10
<211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: GADPH Sense <400> SEQUENCE: 10 tgatgacatc
aagaaggtgg tgaag 25 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 11 <211> LENGTH: 23 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: GADPH Antisense <400>
SEQUENCE: 11 tccttggagg ccatgtaggc cat 23 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 12 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: B7-1 Sense
<400> SEQUENCE: 12 ccttgccgtt acaactctcc 20 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 13 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: B7-1
Antisense <400> SEQUENCE: 13 cggaagcaaa gcaggtaatc 20
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 14
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: B7-2 Sense <400> SEQUENCE: 14 tctcagatgc
tgtttccgtg 20 <200> SEQUENCE CHARACTERISTICS: <210> SEQ
ID NO 15 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: B7-2 Antisense <400> SEQUENCE: 15
ggttcactga agttggcgat 20 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 16 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: OX-2 Sense <400> SEQUENCE: 16
gtggaagtgg tgacccagga 20 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 17 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: OX-2 Antisense <400> SEQUENCE:
17 atagagagta aggcaagctg 20 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 18 <211> LENGTH: 825 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 18
gtgatcagga tgcccttctc tcatctctcc tcctacagcc tggtttgggt catggcagca
60 gtggtgctgt gcacagcaca agtgcaagtg gtgacccagg atgaaagaga
gcagctgtac 120 acacctgctt ccttaaaatg ctctctgcaa aatgcccagg
aagccctcat tgtgacatgg 180 cagaaaaaga aagctgtaag cccagaaaac
atggtcacct tcagcgagaa ccatggggtg 240 gtgatccagc ctgcctataa
ggacaagata aacattaccc agctgggact ccaaaactca 300 accatcacct
tctggaatat caccctggag gatgaagggt gttacatgtg tctcttcaat 360
acctttggtt ttgggaagat ctcaggaacg gcctgcctca ccgtctatgt acagcccata
420 gtatcccttc actacaaatt ctctgaagac cacctaaata tcacttgctc
tgccactgcc 480 cgcccagccc ccatggtctt ctggaaggtc cctcggtcag
ggattgaaaa tagtacagtg 540 actctgtctc acccaaatgg gaccacgtct
gttaccagca tcctccatat caaagaccct 600 aagaatcagg tggggaagga
ggtgatctgc caggtgctgc acctggggac tgtgaccgac 660 tttaagcaaa
ccgtcaacaa aggatattgg ttttcagttc cgctattgct aagcattgtt 720
tccctggtaa ttcttctcat cctaatctca atcttactgt actggaaacg tcaccggaat
780 caggaccgag gtgaattgtc acagggagtt caaaaaatga cataa 825
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 19
<211> LENGTH: 274 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 19 Val Ile Arg Met Pro Phe Ser
His Leu Ser Thr Tyr Ser Leu Val Trp 1 5 10 15 Val Met Ala Ala Val
Val Leu Cys Thr Ala Gln Val Gln Val Val Thr 20 25 30 Gln Asp Glu
Arg Glu Gln Leu Tyr Thr Thr Ala Ser Leu Lys Cys Ser 35 40 45 Leu
Gln Asn Ala Gln Glu Ala Leu Ile Val Thr Trp Gln Lys Lys Lys 50 55
60 Ala Val Ser Pro Glu Asn Met Val Thr Phe Ser Glu Asn His Gly Val
65 70 75 80 Val Ile Gln Pro Ala Tyr Lys Asp Lys Ile Asn Ile Thr Gln
Leu Gly 85 90 95 Leu Gln Asn Ser Thr Ile Thr Phe Trp Asn Ile Thr
Leu Glu Asp Glu 100 105 110 Gly Cys Tyr Met Cys Leu Phe Asn Thr Phe
Gly Phe Gly Lys Ile Ser 115 120 125 Gly Thr Ala Cys Leu Thr Val Tyr
Val Gln Pro Ile Val Ser Leu His 130 135 140 Tyr Lys Phe Ser Glu Asp
His Leu Asn Ile Thr Cys Ser Ala Thr Ala 145 150 155 160 Arg Pro Ala
Pro Met Val Phe Trp Lys Val Pro Arg Ser Gly Ile Glu 165 170 175 Asn
Ser Thr Val Thr Leu Ser His Pro Asn Gly Thr Thr Ser Val Thr 180 185
190 Ser Ile Leu His Ile Lys Asp Pro Lys Asn Gln Val Gly Lys Glu Val
195 200 205 Ile Cys Gln Val Leu His Leu Gly Thr Val Thr Asp Phe Lys
Gln Thr 210 215 220 Val Asn Lys Gly Tyr Trp Phe Ser Val Pro Leu Leu
Leu Ser Ile Val 225 230 235 240 Ser Leu Val Ile Leu Leu Val Leu Ile
Ser Ile Leu Leu Tyr Trp Lys 245 250 255 Arg His Arg Asn Gln Asp Arg
Gly Glu Leu Ser Gln Gly Val Gln Lys 260 265 270 Met Thr <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 20 <211>
LENGTH: 837 <212> TYPE: DNA <213> ORGANISM: Rattus
norvegicus <400> SEQUENCE: 20 atgggcagtc cggtattcag
gagacctttc tgccatctgt ccacctacag cctgctctgg 60 gccatagcag
cagtagcgct gagcacagct caagtggaag tggtgaccca ggatgaaaga 120
aagctgctgc acacaactgc atccttacgc tgttctctaa aaacaaccca ggaacccttg
180 attgtgacat ggcagaaaaa gaaagccgta ggcccagaaa acatggtcac
ttacagcaaa 240 gcccatgggg ttgtcattca gcccacctac aaagacagga
taaacatcac tgagctggga 300 ctcttgaaca caagcatcac cttctggaac
acaaccctgg atgatgaggg ttgctacatg 360 tgtctcttca acatgtttgg
atctgggaag gtctctggga cagcttgcct tactctctat 420 gtacagccca
tagtacacct tcactacaac tattttgaag accacctaaa catcacgtgc 480
tctgcaactg cccgcccagc ccctgccatc tcctggaagg gcactgggtc aggaattgag
540 aatagtactg agagtcactc ccattcaaat gggactacat ctgtcaccag
catcctccgg 600 gtcaaagacc ccaaaactca ggttggaaag gaagtgatct
gccaggtttt atacttgggg 660 aatgtgattg actacaagca gagtctggac
aaaggatttt ggttttcagt cccactgctg 720 ctgagcattg tttctctggt
aattcttctg gtcttgatct ccatcttatt atactggaaa 780 cggcaccgaa
atcaggagcg gggtgagtca tcacagggga tgcaaagaat gaaataa 837 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 21 <211>
LENGTH: 278 <212> TYPE: PRT <213> ORGANISM: Rattus
norvegicus <400> SEQUENCE: 21 Met Gly Ser Pro Val Phe Arg Arg
Pro Phe Cys His Leu Ser Thr Tyr 1 5 10 15 Ser Leu Leu Trp Ala Ile
Ala Ala Val Ala Leu Ser Thr Ala Gln Val 20 25 30 Glu Val Val Thr
Gln Asp Glu Arg Lys Leu Leu His Thr Thr Ala Ser 35 40 45 Leu Arg
Cys Ser Leu Lys Thr Thr Gln Glu Pro Leu Ile Val Thr Trp 50 55 60
Gln Lys Lys Lys Ala Val Gly Pro Glu Asn Met Val Thr Tyr Ser Lys 65
70 75 80 Ala His Gly Val Val Ile Gln Pro Thr Tyr Lys Asp Arg Ile
Asn Ile 85 90 95 Thr Glu Leu Gly Leu Leu Asn Thr Ser Ile Thr Phe
Trp Asn Thr Thr 100 105 110 Leu Asp Asp Gly Gly Cys Tyr Met Cys Leu
Phe Asn Met Phe Gly Ser 115 120 125 Gly Lys Val Ser Gly Thr Ala Cys
Leu Thr Leu Tyr Val Gln Pro Ile 130 135 140 Val His Leu His Tyr Asn
Tyr Phe Glu His His Leu Asn Ile Thr Cys 145 150 155 160 Ser Ala Thr
Ala Arg Pro Ala Pro Ala Ile Ser Trp Lys Gly Thr Gly 165 170 175 Ser
Gly Ile Glu Asn Ser Thr Glu Ser His Ser His Ser Asn Gly Thr 180 185
190 Thr Ser Val Thr Ser Ile Leu Arg Val Lys Asp Pro Lys Thr Gln Val
195 200 205 Gly Lys Glu Val Ile Cys Gln Val Leu Tyr Leu Gly Asn Val
Ile Asp 210 215 220 Tyr Lys Gln Ser Leu Asp Lys Gly Phe Trp Phe Ser
Val Pro Leu Leu 225 230 235 240 Leu Ser Ile Val Ser Leu Val Ile Leu
Leu Val Leu Ile Ser Ile Leu 245 250 255 Leu Tyr Trp Lys Arg His Arg
Asn Gln Glu Arg Gly Glu Ser Ser Gln 260 265 270 Gly Met Gln Arg Met
Lys 275 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO
22 <211> LENGTH: 837 <212> TYPE: DNA <213>
ORGANISM: Mus musculus <400> SEQUENCE: 22 atgggcagtc
tggtattcag gagacctttc tgccatctct ccacctacag cctgatttgg 60
ggcatagcag cagtagcgct gagcacagct caagtggaag tggtgaccca ggatgaaaga
120 aaggcgctgc acacaactgc atccttacga tgttctctaa aaacatccca
ggaacccttg 180 attgtgacat ggcagaaaaa gaaagccgtg agcccagaaa
acatggtcac ctacagcaaa 240 acccatgggg ttgtaatcca gcctgcctac
aaagacagga taaatgtcac agagctggga 300 ctctggaact caagcatcac
cttctggaac acacacattg gagatggagg ctgctacatg 360 tgtctcttca
acacgtttgg ttctcagaag gtctcaggaa cagcttgcct tactctctat 420
gtacagccca tagtacacct tcactacaac tattttgaac accacctaaa catcacttgc
480 tctgcgactg cccgtccagc ccctgccatc acctggaagg gtactgggac
aggaattgag 540 aatagtaccg agagtcactt ccattcaaat gggactacat
ctgtcaccag catcctccgg 600 gtcaaagacc ccaaaactca agttgggaag
gaagtgatct gccaggtttt atacctgggg 660 aatgtgattg actacaagca
gagtctggac aaaggatttt ggttttcagt tccactgttg 720 ctaagcattg
tttctctggt aattcttctg atcttgatct ccatcttact atactggaaa 780
cgtcaccgaa atcaggagcg gggtgaatca tcacagggga tgcaaagaat gaaataa
837
* * * * *